Devices for detecting target biological molecules from cells and viruses

ABSTRACT

Described herein are fluid-manipulation-based devices. Fluid manipulations as described herein can be configured to perform assays on biological samples. In an embodiment, the device includes a reaction chamber, which can includes an integrated sample isolation module, a cell lysis module, a biological target purification module, and an assay mixing module, which can include a microbead with a capture molecule coupled thereto and a nanoparticle having a probe molecule coupled thereto via a label, which can be a spectroscopic label. In an embodiment, the capture and probe molecules can be configured to be coupled together via a biological target to form a biological molecule bead complex. Devices and methods as described herein can manipulate and analyze nanoliter volumes of fluid, microliter volumes of fluid, milliliter volumes of fluid, or greater. Embodiments of the present disclosure can enable random biological assays and rapid, simultaneous analysis of multiple biological samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Ser. No. 62/256,747, having the title “DEVICES FOR DETECTINGTARGET BIOLOGICAL MOLECULES FROM CELLS AND VIRUSES,” filed on Nov. 18,2015, the disclosure of which is incorporated herein in by reference inits entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Contract No.W911SR-12-C-0054 awarded by the US Army Edgewood Chemical and BiologicalCenter (ECBC). The government has certain rights in the invention.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled 222109_1260_patentin_ST25_2.txt, created onJul. 27, 2018. The content of the sequence listing is incorporatedherein in its entirety.

BACKGROUND

Stationary nucleic acid strands have previously been analyzed on a fixedsubstrate, such as a fixed glass slide conventional micro array. Someinstruments that have been used to analyze nucleic acid include CaliperLife Sciences-Perkin Elmer Sciclone NGSx, Zephyr NGS Workstation, andJANUS NGS Express, LabChip GX and LabChip DS, and LabChip XT; IlluminaHiScan and iScan; Nanosphere Verigene System; and Applied Biosystems IonPGM, 3500 Series Genetic Analyzers, and Ion Torrent Sequencing System.

Disadvantages of existing instruments such as those listed above foranalyzing biological agents include: (1) lack of: integrated samplepurification, biomolecule isolation, biomolecule detection, and lack ofsensitivity; (2) long detection times; and (3) high cost of ownership.Additionally, these laboratory instruments are large in size and are notable to be down-scaled to handheld or other portable use in the field.Accordingly, there is a need to address the aforementioned deficienciesand inadequacies.

SUMMARY

Described herein are devices for manipulating fluids, which cancomprise: an assay mixing module configured to mix isolated biologicaltargets with a plurality of microbead complex components contained inone more reagents to generate one or more non-stationary microbeadcomplexes; a flow path in fluidic communication with the assay mixingmodule and configured to receive the one or more non-stationarymicrobead complexes; and an analysis region configured to analyze theone or more non-stationary microbead complexes.

In additional embodiments, the plurality of microbead complex componentsin devices as described herein can further comprise: one or moremicrobeads with one or more capture molecules coupled thereto; one ormore nanoparticles with one or more probe molecules coupled thereto byway of a label; and wherein the capture molecule and probe molecule areconfigured to bind to one or more isolated biological targets.Embodiments of devices as described herein can additionally comprise: abiological target purification module configured to receive products ofcell lysis and isolate biological targets, wherein the biological targetpurification module comprises one or more reagent vessels; and whereinthe biological target purification module is in fluidic communicationwith the assay mixing module and configured to send isolated biologicaltargets to the assay mixing module.

A sample lysis module configured to receive one or more biologicalsamples can also be present in devices as described herein, wherein themodule is configured to lyse cells and create products of cell lysis,the products of cell lysis comprising biological targets, wherein thesample lysis module is configured to send biological targets to thebiological target purification module. Sample lysis modules can beconfigured for thermal or enzymatic lysis.

Further embodiments of devices as described herein can further comprisea sample isolation module containing one or more micro-scale filtersconfigured to isolate biological samples of a desired size, wherein thesample isolation module is in fluidic communication with the cell lysismodule, wherein the sample isolation module is configured to receivesamples containing biological samples from outside of the device andconfigured send biological samples of a desired size to the cell lysismodule. More than one of any of the above modules can be integrated intoa single device or system.

Devices as described herein can comprise one or more valves operativelycoupled to a controller, a computing device, or both, wherein the one ormore valves are configured to direct or restrict flow between themodules of the device, vessels within a module, or both.

Devices as described herein can comprise one or more pumps operationallycoupled to the device and configured to provide positive fluid pressure,negative fluid pressure, or both in the device. One or more pumps asdescribed herein can be operatively coupled to a controller, a computingdevice, or both. One or more pumps as described herein can be syringepumps, dielectrophoresis pumps, solid chemical propellant pumps,individually or in combination.

Fluids of devices as described herein can be femtofluids, picofluids,nanofluids, microfluids, or fluids of a greater volume.

Isolated biological targets as described herein can be nucleic acids.

Labels as described herein can be a spectroscopic dye.

One or more non-stationary microbead complexes as described herein canhave a maximum dimension of a size to fit within a focal point ordiameter of the cross-sectional area of a laser beam configured toanalyze the bead complex.

Microbeads as described herein can be at least two orders of magnitudelarger than the one or more nanoparticles.

One or more microbeads and one or more nanoparticles as described hereincan be within respective separate solutions or are contained within acommon solution.

Devices as described herein can further comprise: at least twomicrobeads having at least two different respective capture moleculescoupled thereto; and at least two nanoparticles having at least twodifferent respective probe molecules coupled thereto via differentrespective labels, the different respective capture molecules anddifferent respective probe molecules configured to be, respectively,coupled together via different respective biological targets to form arandom array of at least two non-statoinary microbead complexes inrandom locations in a fluid.

Labels as described herein can be configured to provide a Raman spectrumto a portable Raman spectrometer.

Capture molecules as described herein can be one or more DNA or RNAmolecules configured to bind to a biological target of a biowarfareagent.

One of the one or more capture molecules and one or more probe moleculesas described herein can be an aptamer, antibody, or nucleic acid.

One or more microbeads as described herein can have multiple capturemolecules coupled thereto, wherein the multiple capture molecules arethe same or different.

Nanoparticles as described herein can be a gold nanoparticle and canhave one or more silver nanoparticles attached thereto.

Described herein are methods of detecting a biological target.Embodiments of methods as described herein can comprise: providing oneor more isolated biological targets; forming one or more non-stationarymicrobead complexes from the one or more isolated biological targets anda plurality of microbead complex components in a volume of fluid; anddetecting the one or more non-stationary microbead complexes with ananalysis platform.

One or more non-stationary microbead complexes used in methods asdescribed herein can comprise: one or more microbeads with one or morecapture molecules coupled thereto; one or more nanoparticles with one ormore probe molecules coupled thereto by way of a label; and wherein thecapture molecule and probe molecule are configured to bind to one ormore isolated biological targets.

Methods as described herein can utilized a volume of fluid being afemtoliter volume, a picoliter volume, a nanoliter volume, or microlitervolume.

Methods as described herein can isolate one or more isolated biologicaltargets from products of cell lysis.

Methods as described herein can lyse one or more biological cells togenerate products of cell lysis, the products of cell lysis comprisingone or more biological targets.

Methods as described herein can process (e.g. separate componentsthereof by size, for example) a biological sample containing one or morebiological cells to isolate the one or more biological cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the present disclosure, asillustrated in the accompanying drawings in which like referencecharacters refer to the same parts throughout the different views. Thedrawings are not necessarily to scale, emphasis instead being placedupon illustrating embodiments of the present disclosure.

FIGS. 1A and 1B illustrate an embodiment biological molecule beadcomplex based on a microbead substrate for surface-enhanced Ramanspectroscopy (SERS) DNA random array assay analysis methods. FIG. 1Bshows an example target sequence (SEQ ID No. 12), an example capturestrand (SEQ ID No. 13), and an example probe strand (SEQ ID No. 14).

FIG. 1C illustrates an embodiment of microbead complex formation andSERS spectra generation according to systems, devices, and methods asdescribed herein.

FIGS. 2A and 2B illustrate an in-line capillary heater for cell lysis.

FIG. 3A is a micrograph illustration of a Bacillus anthracis (BA) sporesolution before heating.

FIG. 3B is a micrograph illustration of the spore solution of FIG. 3Aafter heating.

FIG. 3C is a graph showing a temperature profile of the solution ofFIGS. 3A and 3B during heating and cooling.

FIG. 4 is a graph showing an SERS spectrum of cyanine dye Cy3 detectedfrom a Yersinia pestis (YP) assay.

FIGS. 5A and 5B are graphs illustrating SERS spectra collected forassays of Venezuelan equine encephalitis (VEE) and BA, respectively.

FIG. 6 is a graph illustrating SERS spectra collected from VEE and BAnegative control assays.

FIG. 7A is a graph illustrating SERS measurements on benchtop YP assaysusing a 1 mM YP target.

FIG. 7B is a graph similar to the graph in FIG. 7A, except breadboard YPassays were measured.

FIGS. 8A and 8B are graphs illustrating and SERS measurements on 20randomly selected, fully reacted beads with and without, respectively,the final Cl— treatment. These figures illustrate a strong increase inCy3 SERS signals when the final Cl— treatment is eliminated.

FIG. 9A illustrates a microfluidic channel used to improve thebreadboard for all SERS measurements.

FIG. 9B illustrates a heated mixing chamber that accommodates cellpreparation chemistry to improve the breadboard.

FIG. 10 is a schematic diagram illustrating an expanded breadboardincluding cell lysing and silica gel DNA extraction.

FIG. 11A is a micrograph of a 30 μm diameter glass microbead sampleshowing a position of a 5 μm diameter laser beam for measurement of SERSspectrum.

FIG. 11B is a graph showing and a SERS spectrum obtained from the glassmicrobeads sample of FIG. 11A. The glass microbeads sample includedoligonucleotide functionalized microbeads containing a dye Rhodamine 6G(R6G) (known in the art of spectroscopy) as the probe dye.

FIG. 12 is the graph showing SERS spectrum of the glass microbeadssample containing Cy3 as the probe dye.

FIG. 13 is a graph showing SERS spectrum of a 45 μm diameter glassmicrobeads sample containing oligonucleotide functionalized microbeadsusing Cy3 as the probe dye.

FIGS. 14A and 14B are photographs of an adapter fabricated to testPinpointer™ sensitivity for collecting SERS spectra from assay beads ina channel.

FIG. 15 is a graph showing SERS spectra of a YP assay incorporating theCy3 dye.

FIG. 16A is a photograph showing an embodiment breadboard configurationincluding lysing, DNA purification, and assay stations.

FIG. 16B is a photograph pointing out additional features of thebreadboard of FIG. 16A.

FIG. 17A is a plan view of a 6 inch transparency mask showing single-and sheath-channel designs.

FIG. 17B is a magnified image showing the hydrodynamic focusing observedin a microchannel like those of FIG. 17A with food coloring being pumpedinto the outer (chief) channels, and red food coloring being pumped intothe center channel.

FIG. 18 is a block diagram illustrating an embodiment system fordetecting the target biological molecule.

FIG. 19 is a flow diagram illustrating an embodiment method of detectinga biological target molecule.

FIG. 20 is a schematic diagram illustrating an embodiment kit.

FIG. 21 is a schematic illustration of a random array having microbeadsand nanoparticles configured to attach to different respectivebiological target molecules.

FIG. 22 is a schematic illustration of a random array assay solid statecartridge implementation of an embodiment system.

FIG. 23 is a schematic diagram of a computing device or apparatus 1010which can be coupled to systems as described herein for automatedoperation of the systems.

FIGS. 24A-24D are block diagrams showing embodiments of systems withexamples of module configuration as described herein.

FIGS. 25A-25F are block diagrams demonstrating examples of genericconfigurations of modules of systems as described herein. Biologicalsamples and/or targets can have unidirectional flow in a fluid from theinput, through the sample chamber, to the output.

FIG. 26 is an embodiment of a sample lysis module configured for thermallysis in fluid communication with a biological target purificationmodule configured for DNA extraction as described herein.

FIG. 27 is an embodiment of an assay mixing module configured for SERSdetection of DNA target molecules.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit (unlessthe context clearly dictates otherwise), between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of organic chemistry, inorganic chemistry, Ramanspectroscopy, protein isolation, nucleic acid isolation, antibodyisolation, hybridization, microfluidics, spectroscopy, which are withinthe skill of the art. Such techniques are explained fully in theliterature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is inatmosphere. Standard temperature and pressure are defined as 25° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

Description and Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Additional definitions andclarification of terms used herein follows.

Nucleic Acid Molecules

As used herein, the term “target biological molecule” or “biologicaltarget molecule” refers to a biological molecule, e.g., a nucleic acidmolecule, protein, lipid, carbohydrate, peptide, antibody, or the like,whose presence or absence in a sample (e.g., a biological sample) isdesired to be detected.

As used herein, the term “nucleic acid molecule” refers to a polymercomprising nucleotide monomers (e.g., ribonucleotide monomers ordeoxyribonucleotide monomers). The term “nucleic acid molecule” caninclude, for example, genomic DNA, cDNA, ssDNA, dsDNA, RNA, mRNA, tRNA,shRNA, rRNA, snRNA, miRNA, tmRNA, dsRNA and DNA-RNA hybrid molecules.Nucleic acid molecules can be naturally, e.g., occurring, recombinant,or synthetic. Nucleic acid molecules can be single-stranded,double-stranded or triple-stranded. In some embodiments, nucleic acidmolecules can be modified. Nucleic acid modifications include, forexample, methylation, acetylation, substitution of one or more of thenaturally occurring nucleotides with a nucleotide analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, and thelike), charged linkages (e.g., phosphorothioates, phosphorodithioates,and the like), pendent moieties (e.g., polypeptides), intercalators(e.g., acridine, psoralen, and the like), chelators, alkylators, andmodified linkages (e.g., alpha anomeric nucleic acids, and the like). Ifthe polymer is a double-stranded, “nucleic acid” can refer to either orboth strands of the molecule.

The term “nucleotide” refers to naturally-occurring ribonucleotide ordeoxyribonucleotide monomers, as well as their non-naturally-occurringderivatives and analogs. Nucleotides can include, for example,nucleotides comprising naturally-occurring bases (e.g., adenosine,cytidine, thymidine, guanosine, inosine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotidescomprising modified bases (e.g., 2-aminoadenosine, 2-thiothymidine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine).

As used herein, the term “nucleotide base” refers a heterocyclicnitrogenous base of a nucleotide or nucleotide analog (e.g., purine,pyrimidine, 7-deazapurine). Suitable nucleotide bases can include, butare not limited to, adenine, cytosine, thymine, guanine, uracil,hypoxanthine and 7-deaza-guanine. The base pair can be either aconventional (standard) Watson-Crick base pair or a non-conventional(non-standard) non-Watson-Crick base pair, for example, a Hoogstein basepair or bidentate base pair.

The term “sequence,” in reference to a nucleic acid molecule, refers toa contiguous series of nucleotides that are joined by covalent linkages,such as phosphorus linkages and/or non-phosphorus linkages (e.g.,peptide bonds). A sequence can be read in a 5′→3′ direction or a 3′→5′direction.

The term “target sequence” refers to a sequence, e.g., a nucleotidesequence within a target biological molecule that is capable of forminga hydrogen-bonded duplex with a complementary nucleotide sequence (e.g.,a substantially complementary sequence on a nucleotide probe). A targetsequence can also be an amino acid sequence translated from a nucleicacid sequence.

As used herein, “complementary” refers to sequence complementaritybetween different nucleic acid strands or between regions of the samenucleic acid strand. One region of a nucleic acid is complementary toanother region of the same or a different nucleic acid if, when theregions are arranged in an anti-parallel fashion, at least onenucleotide residue of the first region is capable of base pairing (i.e.,hydrogen bonding) with a residue of the second region, thus forming ahydrogen-bonded duplex.

The term “substantially complementary” refers to two nucleic acidstrands (e.g., a strand of a target nucleic acid molecule and acomplementary single-stranded oligonucleotide probe) that are capable ofbase pairing with one another to form a stable hydrogen-bonded duplexunder stringent hybridization conditions, including the hybridizationconditions described herein. In general, “substantially complementary”refers to two nucleic acid strands having at least 70%, for example,about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%, complementarity.

The term “hybrid” molecule refers to a double-stranded nucleic acidmolecule formed by hydrogen bonding between complementary nucleotides.

Biological Targets

In certain embodiments, the target biological molecule comprises anucleotide sequence of an organism or protein, e.g., a pathogenicmicroorganism, including, but not limited to, a virus (e.g.,Adenoviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Picornaviridae,Polyomavirus, Retroviridae, Rhabdoviridae, Togaviridae), a bacterium(e.g., Bacillus, Bordetella, Borrelia, Brucella, Campylobacter,Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus,Escherichia, Francisella, Haemophilus, Helicobacter, Legionella,Leptspira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseodomonas,Rickettsia, Salmonella, Shigella, Staphylococcus, Steptococcus,Treponema, Vibrio, Yersinia), a fungus (e.g., Aspergillus, Candida,Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys), or a eukaryoticmicroorganism, such as, for example, a protist (e.g., Plasmodium) or ahelminth (e.g., Ascaris). Examples of pathogens include, among others,Ebola virus, Dengue virus, hantaviruses, Lassa fever virus, Marburgvirus, Variola virus, West Nile virus, Venezuelan equine encephalitisvirus (VEE), Eastern equine encephalitis virus, Western equineencephalitis virus, Japanese equine encephalitis virus, Yellow fevervirus, Rift Valley fever virus, Bacillus anthracis (BA), Staphylococcusaureus, Clostridium botulinum, Brucella abortus, Brucella melitensis,Brucella suis, Vibrio cholera, Corynebacterium diphtheria, Shigelladysenteriae, Escherichia coli, Burkholderia mallei, Listeriamonocytogenes, Burkholderia pseudomallei, Yersinia pestis (YP), andFrancisella tularensis. In some embodiments, the organism is abiological warfare agent (BWA). In some embodiments, the target moleculeis from a spore. In other embodiments, the target biological molecule isa prion. Prions play a role in the etiology of bovine spongiformencephalopathy, Creutzfeldt-Jakob Disease, variant Creutzfeldt-JakobDisease, Gerstmann-Sträussler-Scheinker syndrome, Fatal FamilialInsomnia and kuru. In additional embodiments, the target biologicalmolecule is a protein, a peptide, lipid, carbohydrate, or antibody.

In some embodiments, the target molecule is non-pathogenic, for example,a nucleic acid a host produces, e.g., during infection.

In certain embodiments, the target biological molecule comprises anucleotide sequence that is at least about 70%, about 80%, about 90%,about 95%, about 98%, about 99% or about 100% identical to a wild-typenucleotide sequence of an organism. In some embodiments, the targetbiological molecule comprises a nucleotide sequence that is 100%identical to a nucleotide sequence of an organism.

As used herein, “sequence identity” means that two nucleotide or aminoacid sequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 70% sequence identity,or at least 80% sequence identity, or at least 85% sequence identity, orat least 90% sequence identity, or at least 95% sequence identity ormore. For sequence comparison, typically one sequence acts as areference sequence (e.g., parent sequence), to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm described in Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm described inNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method described in Pearson & Lipman, Proc. Nat'l. Acad. Sci.USA 85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byvisual inspection (see generally Ausubel et al., Current Protocols inMolecular Biology), all of which are incorporated by reference in theirentirety. One example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al., J. Mol. Biol. 215:403(1990). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (accessiblethrough the National Institutes of Health NCBI internet server). Defaultprogram parameters can typically be used to perform the sequencecomparison, although customized parameters can also be used. For aminoacid sequences, the BLASTP program uses as defaults a wordlength (W) of3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

Biological Samples

The term “biological sample” refers to a material of biological origin(e.g., cells, tissues, organs, bodily fluids, or an environmentalsource). Biological samples can comprise, for example, nucleic acid(e.g., DNA or RNA) extracts, cell lysates, whole cells, proteinextracts, tissues (including tissue biopsies), organs, lipid extracts,and bodily fluids (e.g., blood, wound fluid, plasma, serum, spinalfluid, lymph fluid, tears, saliva, mucus, sputum, urine, semen, amnioticfluid). A biological sample can be obtained from any of a variety ofsuitable sources. In one embodiment, the biological sample is obtainedfrom a subject (e.g., a human, a non-human mammal, a patient). In aparticular embodiment, the biological sample is obtained from a human.

In some embodiments, a biological sample can be obtained from anenvironmental source, such as air, water, or soil.

Probes and Labels

As used herein, the term “probe” refers to a nucleic acid molecule thatincludes a target-binding region that is substantially complementary toa target sequence in a target nucleic acid and, thus, is capable offorming a hydrogen-bonded duplex with the target nucleic acid.Typically, the probe is a single-stranded probe and can have one or moredetectable labels to permit the detection of the probe followinghybridization to its complementary target. As used herein,“target-binding region” refers to a portion of a probe that is capableof forming a hydrogen-bonded duplex with a complementary target nucleicacid.

In a particular embodiment, the probes used in the present disclosureare oligonucleotide probes (e.g., single-stranded DNA oligonucleotideprobes). Typical oligonucleotide probes are linear and range in sizefrom about 6 to about 100 nucleotides, preferably, about 20 to about 50nucleotides. In a particular embodiment, the oligonucleotide probes areabout 30 nucleotides in length.

In some embodiments, suitable probes for use in the methods of thepresent disclosure include, but are not limited to, DNA probes, RNAprobes, peptide nucleic acid (PNA) probes, locked nucleic acid (LNA)probes, morpholino probes, glycol nucleic acid (GNA) probes and threosenucleic acids (TNA) probes. Such probes can be chemically orbiochemically modified and/or may contain non-natural or derivatizednucleotide bases. For example, a probe may contain modified nucleotideshaving modified bases (e.g., 5-methyl cytosine) and/or modified sugargroups (e.g., 2′O-methyl ribosyl, 2′O-methoxyethyl ribosyl, 2′-fluororibosyl, 2′-amino ribosyl). In some embodiments, useful probes can belinear, circular or branched and/or include domains capable of formingstable secondary structures (e.g., stem- and loop and loop-stem-loophairpin structures). In one embodiment, linear probes are used. Methodsof producing probes useful in the methods of the present disclosure arewell known in the art and include, for example, biochemical,recombinant, synthetic and semi-synthetic methods. (see, e.g., Glick andPasternak, Molecular Biotechnology: Principles and Applications ofRecombinant DNA (ASM Press 1998)). For example, solution or solid-phasetechniques can be used.

Probes useful in the methods of the present disclosure can furthercomprise one or more labels (e.g., detectable labels). Labels suitablefor use according to the present disclosure are known in the art andgenerally include any molecule that, by its nature, and whether bydirect or indirect means, provides an identifiable signal allowingdetection of the probe.

The term “label,” as used herein, refers to a moiety that indicates thepresence of a corresponding molecule (e.g., a probe molecule) to whichit is bound. In some embodiments, the label is attached to the moleculevia a linker, crosslinker, or spacer.

A “linker,” in the context of attachment of two molecules (whethermonomeric or polymeric), means a molecule (whether monomeric orpolymeric) that is interposed between and adjacent to the two moleculesbeing attached. A “linker” can be used to attach, e.g., probe moleculeand a label (e.g., a detectable label, e.g., a spectroscopic dye). Thelinker can be a nucleotide linker (i.e., a sequence of the nucleic acidthat is between and adjacent to the non-adjacent sequences) or anon-nucleotide linker.

Probe labeling can be performed using standard laboratory techniquesknown in the art, e.g., during synthesis or, alternatively,post-synthetically, for example, using 5′-end labeling. Labels can beadded to the 5′, 3′, or both ends of the probe (see, e.g., U.S. Pat. No.5,082,830), or at base positions internal to the oligonucleotide.

In a particular embodiment, the probes employed in the presentdisclosure include one or more Raman labels. “Raman label” or“Raman-active label” as used herein, is any substance which produces adetectable Raman spectrum, which is distinguishable from the Ramanspectra of other components present, when illuminated with a radiationof the proper wavelength. Other terms for a Raman label include “Ramandye” and “Raman reporter molecule.”

A variety of suitable Raman labels are known in the art, including, butnot limited to, 4-(4-Aminophenylazo)phenylarsonic acid monosodium salt,arsenazo I, basic fuchsin, Chicago sky blue, direct red 81, disperseorange 3, HABA (2-(4-hydroxyphenylazo)-benzoic acid), erythrosin B,trypan blue, ponceau S, ponceau SS, 1,5-difluoro-2,4-dinitrobenzene,cresyl violet and p-dimethylaminoazobenzene. In some embodiments, thelabels can be any of those described herein. In some embodiments, thelabel is a cyanine dye such as Cy3, Cy3.5, Cy5, Cy7, and Cy7.5. In someembodiments, the dye is Rhodamine 6G.

One or more Raman labels may be bound to a particle (e.g., nanoparticle,such as a gold nanoparticle). For particle-based detection probes, theRaman labels or dyes can be attached directly or indirectly to theparticle. The Raman label can be modified with a functional group, e.g.,a thiol, amine, or phosphine that can bind to the surface of theparticle such as a metallic nanoparticle. If desired, the Raman dye canbe further functionalized with a molecule such as an oligonucleotide(e.g., polyadenosine, polythymidine) for enhanced nanoparticle stabilityor with a specific binding pair member (such as an oligonucleotidehaving a sequence that is complementary to at least a portion of anucleic acid target or a receptor for a particular ligand).Alternatively, the Raman label can be conjugated with a molecule or anylinker, e.g., polyA or polyT oligonucleotide, that bears a functionalgroup for binding to the particle. The polyA or polyT oligonucleotide towhich the Raman labels are conjugated is not complementary to any targetnucleic acid.

Nucleic Acid Hybridization

The term “hybridization conditions” refers to hybridization conditionsthat affect the stability of hybrids, e.g., temperature, saltconcentration, pH, formamide concentration, incubation time, and thelike. These conditions can be empirically optimized to maximize specificbinding, and minimize nonspecific binding, of a probe to a targetnucleic acid. The term “stringent hybridization conditions” refers tohybridization conditions under which complementary (e.g., substantiallycomplementary) nucleic acids specifically hybridize with one another. Insome embodiments, the conditions are isothermal.

Generally, hybridization is performed under conditions sufficient for aprobe to hybridize with a complementary target nucleic acid in abiological sample. Suitable hybridization buffers and conditions for insitu hybridization techniques are generally known in the art. (See,e.g., Sambrook and Russell, supra; Ausubel et al., supra. See alsoTijssen, Laboratory Techniques in Biochemistry and Molecular Biology,Vol. 24: Hybridization with Nucleic Acid Probes (Elsevier, N.Y. 1993)).

Optimal hybridization conditions for a given target sequence and itscomplementary probe will depend upon several factors such as saltconcentration, incubation time, and probe concentration, composition,circumstances of use and length, as will be appreciated by those ofordinary skill in the art. Based on these and other known factors,suitable binding conditions can be readily determined by one of ordinaryskill in the art and, if desired, optimized for use in accordance withthe present systems and methods. Typically, hybridization is carried outunder stringent conditions that allow specific binding of substantiallycomplementary nucleotide sequences. Stringency can be increased ordecreased to specifically detect target nucleic acids having 100%complementarity or to detect related nucleotide sequences having lessthan 100% complementarity (e.g., about 70% complementarity, about 80%complementarity, about 90% complementarity). Factors such as, forexample, the length and nature (DNA, RNA, base composition) of the probesequence, nature of the target nucleotide sequence (DNA, RNA, basecomposition, presence in solution or immobilization) and theconcentration of salts and other components in the hybridization buffer(e.g., the concentration of formamide, dextran sulfate, polyethyleneglycol and/or salt) in the hybridization buffer/solution can be variedto generate conditions of either low, medium, or high stringency. Theseconditions can be varied based on the above factors, either empiricallyor based on formulas for determining such variation (see, e.g., Sambrooket al., supra; Ausubel et al., supra).

Washes are performed in a solution of appropriate stringency to removeunbound and/or non-specifically bound probes. An appropriate stringencycan be determined by washing the sample in successively higherstringency solutions and reading the signal intensity between each wash.Analysis of the data sets in this manner can reveal a wash stringencyabove which the hybridization pattern is not appreciably altered andwhich provides adequate signal for the particular probes of interest.

Suitable wash buffers for in situ hybridization methods are generallyknown in the art (See, e.g., Sambrook and Russell, supra; Ausubel etal., supra. See also Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 24: Hybridization with Nucleic Acid Probes(Elsevier, N.Y. 1993)). Wash buffers typically include, for example, oneor more salts (e.g., sodium salts, lithium salts, potassium salts) andone or more detergents (e.g., an ionic detergent, a non-ionicdetergent). Suitable detergents for a wash buffer include, but are notlimited to, sodium dodecyl sulfate (SDS), Triton® X-100, Tween® 20,NP-40, or Igepal® CA-630. De-ionized water may also be used as a washsolution.

Breadboard

A breadboard is a board for making electrical or experimental electricalcircuits. Systems as described herein can be constructed on abreadboard, be part of a breadboard, a breadboard can be part of asystem, and in some embodiments the system can be referred to as a“breadboard”

Nanoparticle Complex

As used herein, nanoparticle complex refers to a nanoparticle coupled toone or more probe molecules via a dye. The one or more probe moleculescan be identical in sequence or structure, or can be different. One ormore probe molecules can be configured by an end-user according to oneor more desired target molecules. The dye used can additionally beconfigured by an end-user depending on the analysis method.

Microbead Complex

As used herein, microbead complex refers to a microbead coupled to oneor more capture molecules. The one or more capture molecules can beidentical in sequence or structure, or can be different. One or morecapture molecules can be configured by an end-user according to one ormore desired target molecules.

A microbead complex as used herein can also refer to a microbead coupledto one or more capture molecules, hybridized to a target molecule, andthe target molecule can further be hybridized to a nanoparticle with oneor more probe molecules couple to the nanoparticle via one or more dyes.

DISCUSSION

Described herein are methods and devices for manipulating and analyzingfluids containing biological targets. Devices and methods as describedherein can prepare analytical assays for biological targets utilizingmicrobeads and/or nanoparticles in a non-stationary phase, and candetect or analyze biological targets from these assays quickly. Devicesand methods as described herein can comprise integrated systemsutilizing more than one of a sample isolation module, a cell lysismodule, a biological target purification module, an assay mixing module,a flow path, and an analysis region. Devices and methods as describedherein can be comprised of microfluidic components configured forfemto-, pico-, nano- and micro-liter volumes.

Embodiments of the present disclosure can analyze biological materialsamples obtained from bulk, body fluids, or environmental sources, forexample, by detecting and identifying biological target molecules (e.g.,nucleic acid) from the biological material samples. Environmentalsources can include air, soil, or water, for example. Biological samplescan include a sample from a cell, bacterium, virus, fungus, spore, andvirus for example. Embodiments also allow for detection of biomarkers,which can include non-pathogen target molecules, such as DNA, RNA,lipid, carbohydrate, antibodies, or protein that a human or animal hostproduces during infection by a pathogen. From a sample of biologicalmaterial, in some embodiments devices can lyse the cells, extract andpurify cell DNA, capture and label the DNA onto microbeads, and thendetect and identify the DNA using surface enhanced Raman spectroscopy(SERS), for example. Embodiments can make use of SERS labels, randomarray assays, and SERS detection. Embodiments are scalable to handheldor other portable sensor platforms, such as a platform configured foruse in a vehicle. Embodiment methods can enable a biowarfare agent(BWA), microorganism, cell, bacteria, virus, spore, or fungus, forexample, to be analyzed quickly by simply injecting a biological sample,pressing a button on a device, and obtaining analysis results within aslittle as 30 minutes, for example. Thus, embodiments can be much fasterthan and just as sensitive as existing laboratory-based devices.

Advantages of embodiments of the present disclosure can include fasthybridization kinetics for fast assay formation and rapid detection ofbiological targets, such as those present in biological warfare agents,an ability to analyze multiple assays in a single cartridgesimultaneously, and an ability to obtain SERS readouts with a fixedoptical configuration. Furthermore, embodiments of the presentdisclosure can be highly reliable for field use, with no moving parts.Test cartridges can also be small, scaling only a volume of microscopicbead complexes to add additional assays.

Described herein are embodiments of systems and corresponding methods,for detecting one or more target biological molecules. Systems (e.g.,apparatuses, devices, analysis systems, analysis devices, targetanalysis devices) are described herein that can detect a biologicaltarget molecule. A system can be comprised of one or moreinterchangeable modules (as used herein, a module can also refer to achamber). Modules used in a system or systems as described herein, canbe integrated into a single system or mobile cartridge and configuredfor sample isolation, sample lysis, target purification, target assays,and target flow. In embodiments of the present disclosure, systems asdescribed herein can include a reaction chamber (a reaction chamber caninclude any one or more of a sample isolation module, a cell lysismodule, a biological target purification module, and an assay mixingmodule) that can be configured to enable formation of a biologicalmolecule bead complex, a flow path (interchangeably referred to hereinas a channel) configured to enable transport of the bead complex fromthe reaction chamber to an analysis region, and a spectrometerconfigured to analyze the bead complex at the analysis region. Systemsas described herein can be configured to handle, process, and transportfluids on a microfluid scale (microliter volumes). Parts, or modules, ofthe system can be self-contained in a cartridge with a small footprintthat is suitable for field use.

Systems as described herein can have a reaction chamber. A reactionchamber can comprise one or modules, and the modules can be sampleisolation modules, cell lysis modules, biological target purificationmodules, assay mixing modules, flow modules, or others, as describedherein. Modules within the reaction chamber can be in fluidiccommunication with each other. A reaction chamber can contain more thanone type of a certain module. Modules can be connected in series or inparallel, or both, and one skilled in the art could configure a reactionchamber according to the present disclosure according to a givenapplication or desired analysis target[s]. A reaction chamber asdescribed herein can comprise a sample lysis module and/or a targetpurification module. In some embodiments, a reaction chamberadditionally can include a sample isolation module.

A reaction chamber can isolate biological samples and can contain asample isolation module. As described herein, a module for sampleisolation can be a sample isolation module. In an embodiment, a fluid(air or water) containing a biological sample can flow into a sampleisolation module, and the fluid can flow through one or more filters inthe sample isolation module to isolate biological samples of a desiredsize. A sample isolation module can be configured to isolate biologicalsamples of a desired size using one or more filters, and sizes offilters and number of filters can be adjusted according to the desiredbiological sample of interest. Examples of filters that can be used canbe 20 μm filters, 2 μm filters, and 0.22 μm filters. If more than onefilter is used in module, the filters can be connected in fluidiccommunication in series and/or in parallel. A sample isolation modulecan be connected in fluidic communication with one or more additionalmodules within the system in series and/or in parallel. A sampleisolation module can further comprise a sample inlet, allowing for inputof samples containing biological samples into the system by a devicesuch as a pipette or a syringe. An example of a sample inlet can be aninjection port in a valve, or a rubber gasket as used in commonneedle-based clinical blood collection tubes.

A reaction chamber can lyse cells and can contain a cell lysis module.As described herein, a module for cell lysis can be a cell lysis module.A cell lysis module can lyse cells and/or fragment cellular components,such as nucleic acids, lipids, carbohydrates, and proteins. A cell lysismodule can be comprised of a chamber where one or more biologicalsamples are lysed. In an embodiment, a cell lysis module is configuredfor thermal cell lysis. For example, a cell lysis module for thermalcell lysis can be comprised of a capillary tube (which can be thechamber in this embodiment) coupled to one or more heating elements,wherein one or more biological samples within the tube are heated by theone or more heating elements. The one or more heating elements can beoperated manually or can be electrically couple to a controller and/or acomputing device for automated operation. The cell lysis module can bein fluid communication with a sample isolation module, connected inseries. A system can comprise more than one sample isolation module andmore than one cell lysis module, and the modules can be in configuredfor series and/or parallel fluid communication between modules dependingon a desired application.

In another embodiment, a cell lysis module can be configured forenzymatic cell lysis. A cell lysis module configured for enzymatic celllysis can comprise a chamber or an area where one or more cells can bemixed with a buffer and/or lysis enzyme (for example a lysozyme). A celllysis module can configured for enzymatic cell lysis can additionallycomprise one or more reagent chambers in fluidic communication with themixing chamber or area, that store components used for enzymatic celllysis. One or more fluid pumps can be used to mix the components (suchas cells, buffers, and/or enzymes) within the module. Otherconfigurations of cell lysis modules can be realized according to othercell lysis protocols known in the art (i.e., mechanical methods such asvortexing or other methods, such as sonication). In theseconfigurations, a cell lysis module can comprise a sample chamber orarea coupled to a mechanical lysis element. A cell lysis module asdescribed herein could utilize more than one type of cellular lysis, forexample could comprise a heating element in addition to a mechanicallysis element. One skilled in the art could configure a cell lysismodule according to a desired cell type or application. A cell lysismodule can further comprise a sample inlet, allowing for input ofbiological samples into the system by a device such as a pipette or asyringe. An example of a sample inlet can be an injection port in avalve, a rubber gasket as used in common needle-based clinical bloodcollection tubes, and/or a receiving tube.

Cell lysing modules can additionally be configured to lyse a cell torelease the target DNA molecule. The cell lysing chamber can be coupledto the reaction chamber via one or more valves, and the system can alsoinclude a controller operatively coupled to the one or more valves tocause the valves to open and close in the sequence enabling the targetDNA molecule to flow from the cell lysing chamber to the reactionchamber.

A reaction chamber can isolate or purify biological targets frombiological samples and can contain a biological target purificationmodule. As described herein, a module for biological target purificationand/or isolation can be a biological target purification module (alsoreferred to herein as biological target isolation modules). A biologicaltarget purification module can be configured to receive components ofbiological samples from one or more cell lysis modules. A biologicaltarget isolation module can be in fluidic communication with one or morecell lysis modules, in series and/or parallel. Biological targetisolation modules as described herein can have a chamber, area, ordevice in or on which biological targets are isolated. Biological targetisolation modules can further comprise one or more reagent chambers orvessels (which can be configured to receive reagents) where one or morereagents are stored, and the chambers can be in fluidic communicationwith an area, chamber, or part of the module where purification takesplace. One or more pumps can be used to mix biological sample componentsand reagents that are stored within the biological target purificationmodule. In an embodiment, biological targets are isolated on one or moresilica gel membranes or capillaries.

A biological target purification module can be configured forpurification or isolation of components of biological samples orcomponents of cells, for example any one or more of DNA, RNA, lipid,carbohydrate, protein, antibody, etc., individually or in combination.Isolation of these components is well known in the art, and one skilledin the art would be able to configure a biological target purificationmodule according to a desired biological target of interest. Operationof the module can be performed by a user manually according towell-known steps of target isolation in the art. Additionally, operationof the module can be automated with the assistance of a pumps, valves,controller and/or computing device (individually or in combination)configured or programmed via to send instructions that would allow themodule to perform purification steps known in the art. A biologicaltarget purification module can also be configured to send purified (forexample about 75%- to 100% pure target) and/or isolated targets to othermodules, such as an assay mixing module, and can be in fluidiccommunication with other modules. A biological target purificationmodule can also comprise heating or cooling elements, to heat or coolmaterial therein.

FIG. 26 shows an embodiment of a biological target purification modulefor nucleic acid purification, with a configuration based on acommercially available DNeasy® kit from the vendor Qiagen and associatedreaction protocol. A silica gel capillary is present for nucleic acidisolation (although other devices with one or more silica membranes canbe used), the module is configured to hold reagents according to the kitin separate reagent chambers or vessels, and the specific fluidicconfiguration of the module has been realized according to steps of thekit. In the figure, electrical connections between valves and acontroller are shown, and the controller and/or a computing device canbe programmed to carry out the steps laid out in the kit automaticallywith the assistance of one or more pumps (not shown). Operation of themodule can also be performed manually by a user. Optional opticalsensors are also shown in the example, which can be used by thecontroller and/or computing device for feedback loops that can monitor,start fluid flow, and/or actively regulate fluid flow in real-time asthe target purification assay commences. Opening and closing ofdifferent valves as shown can be down manually by a user orautomatically according to steps in the instruction manual of the kit.Optional buffer loops are shown as well which can be utilized to clearfluid lines or wash the silica between uses. Based on the exampleembodiment shown in FIG. 26, one skilled in the art could modify themodule for other kits or to perform isolation of targets other thannucleic acids.

A reaction chamber can prepare biological targets for detection and cancontain an assay mixing module. As described herein, a module for assaymixing can be an assay mixing module or an assay chamber. An assaymixing module can comprise a region, area, or chamber within whichisolated biological targets can be mixed with detection assaycomponents, such as labels for detection. An assay mixing module can bein fluid communication with a biological target purification module, andbe configured to receive isolated or purified biological targets fromthe assay biological target purification module. An assay mixing modulecan further comprise a thermal cycler for temperature cycling to aid inmixing and binding of target biological samples to reagent components.

An assay mixing module can comprise one or more reagent vessels. Anassay mixing module can comprise one or more reagent or buffer loops.Reagents in the assay mixing module can comprise components that canbind to biological sample targets. Reagent components can comprise beads(which be glass), capture molecules (which can be short peptides oroligonucleotides of predetermined sequence, for example), probemolecules (which can be short peptides or oligonucleotides ofpredetermined sequence, for example), nanoparticles, dyes, silverparticles, gold particles, phosphate buffered saline, nitrate buffer,silver solution, and an optional chloride solution. Valves can be usedin the module to direct or restrict flow of reagents. Pumps can beutilized to drive fluid flow in the module. Valves and pumps can beoperated manually, or can be electrically coupled to a controller and/ora computing device programmed to send instructions to operate the moduleaccording to a detection assay reaction protocol. The module can beoperated manually, automated with the assistance of a controller and/ora computing device, or a combination thereof. Reagent vessels and/orloops can be configured to receive liquid or air, by way of a syringe,pipette, micropipette, or simply by pouring liquid in. An example of anembodiment of an assay mixing chamber is show in FIG. 27.

In an embodiment, the assay mixing module can comprise: (1) one or moremicrobeads with a capture molecule coupled thereto, and (2) one or morenanoparticles having a probe molecule coupled thereto via a label. Thecapture molecule and probe molecule can be coupled together via abiological target to form a biological molecule bead complex, and theassay mixing module can be configured to couple or hybridize thesecomponents. Examples of coupled or hybridized bead complexes are shownin FIG. 1A and FIG. 1B. FIG. 1B shows an example target sequence (SEQ IDNo. 12), an example capture strand (SEQ ID No. 13), and an example probestrand (SEQ ID No. 14). These components can be present as compositionsin one or more reagent solutions present in one or more reagent vesselsor loops. Microbead compositions and nanoparticle compositions can bepresent in solutions as individual reagents, or can be present in onemixed reagent.

The biological target that can be coupled to or hybridized with theabove microbeads and nanoparticles can be a nucleic acid (e.g., DNA orRNA) molecule. The label can be a spectroscopic dye for Ramanspectroscopic analysis or other analysis (fluorescence, for example).The biological molecule bead complex can be of a size to fit within afocal point or diameter of a cross-section of a laser beam that can beconfigured to analyze the bead complex. The microbead can be at leasttwo orders of magnitude larger than the nanoparticles. The microbead andnanoparticles can be within corresponding collections of multiplemicrobeads and multiple nanoparticles in respective separate solutionsor a common solution.

The assay mixing module can also include at least two microbeads havingdifferent respective capture molecules coupled thereto and at least twonanoparticles having a different respective probe molecules coupledthereto via different respective labels. The different respectivecapture molecules and different respective probe molecules can be,respectively, coupled together via different respective biologicaltargets to form a random array of at least two biological molecule beadcomplexes in random locations in a fluid.

The label can be configured to provide a Raman spectrum to a portableRaman spectrometer. In some embodiments, the capture molecule can be anucleic acid (e.g., DNA or RNA) molecule configured to bind to thebiowarfare agent or component thereof. At least one of the capturemolecule and probe molecule can be an aptamer, antibody, or DNAmolecule. The microbeads can have multiple capture molecules coupledthereto, and the multiple capture molecules can be the same. Themicrobead can have multiple capture molecules coupled thereto, and themultiple capture molecules can be different. The nanoparticles can begold nanoparticles and can have one or more silver nanoparticlesattached thereto. Further description can be found in the examplesection below.

An assay mixing module or reaction chamber can form a bead complex ormolecular bead complex. The biological molecule bead complex can beconfigured to be coupled to a target nucleic acid molecule present in abiological sample. The bead complex can be a DNA bead complex and caninclude a microbead with a biological capture molecule coupled theretoand a nanoparticle coupled to a biological probe molecule via a label.The reaction chamber can be configured to facilitate coupling of thecapture molecule and probe molecule together through a binding reactionwith a target biological molecule to form the bead complex. In someembodiments, biological capture and probe molecules can include DNA,RNA, proteins, aptamers, or antibodies. Molecules coupled to microbeadsand nanoparticles can be nucleic acids of a pre-determined sequence. Thepre-determined sequence can be a complementary nucleic acid sequence toa nucleic acid sequence present. The nucleic acid sequence present in abiological sample, can be nucleic acid of a cell or organism, or forexample DNA of a biological warfare agent such as bacillus anthracis.

An assay mixing module can additionally comprise an air or gas sourceand/or element to bubble components of a detection assay that are mixedor to separate reagents before mixing. Example 14 below contains someexamples of assays and protocols that can be embodied in an assay mixingmodule of systems described herein.

The label can be a spectroscopic dye, which can have an absorption bandat a wavelength of the laser configured to illuminate the bead complexat the analysis region. A matching of the wavelength of the laser withthe absorption band can be used to enhance the strength of a signalproduced by the molecule bead complex greatly through surface enhancedresonance Raman spectroscopy, for example. Absorption wavelengths ofdyes and emission wavelengths of lasers are well known in the art, andone skilled in the art would be able to choose a suitable dye and laserbased on the absorption wavelength of the dye and emission wavelength ofthe laser. It can additionally be possible to multiplex dyes, and havebead complexes with dyes having different absorption wavelength or acomposition of bead complexes with different dyes having differentabsorption wavelengths.

The spectrometer can be a Raman spectrometer and can be configured tocause the bead complex to react to the surface enhanced resonance Ramanspectroscopy stimulation signal, such as a laser beam. The spectrometer,alternatively, can be configured to perform fluorescence spectroscopy toanalyze the bead complex and can be configured to analyze, at theanalysis region, a plurality of bead complexes having differentrespective, associated target biological molecules.

Systems as described herein can also have a one or more flow paths thatdirect or transport labeled biological targets from the assay mixingmodule or reaction chamber to one or more analysis platforms or analysisregions. The flow path can be a channel of a microfluidic device, andthe analysis region can be a part of a microfluidic device containing aflow path. Transporting the bead complex through the flow path caninclude transporting the complex through a microfluidic channel. Thebead complex containing one or more microbead complexes can be in afluid or aqueous suspension, and can be non-stationary.

Valves (2-way, 3-way, or more depending on the configuration of themodule) can be used to direct or restrict flow within the systems or oneor more modules of systems of the present disclosure (to the filters,away from the filters, to another module, etc.). Valves can becontrolled manually or can be electrically (aka operatively) connectedto a controller and/or a computing device as described herein andcontrol electronically (i.e. in an automated system).

Differences or changes in fluid pressure (positive or negative, morepositive or more negative, etc.) can drive fluid through the systemand/or through individual modules of the system. Pressure changes thatdrive fluid can be derived from one or more pumps. One or more pumps asdescribed herein can be in fluid communication with systems as describedherein and connected to a system or to individual modules of a system inseries or in parallel. One or more pumps as described herein can beoperatively coupled to or integrated with a reaction chamber, one ormore modules of a reaction chamber, one or more reagent vessels, one ormore reagent loops, or any other components of the system which requiresthe flow or mixing of fluid. One or more pumps can be integrated intoreagent chambers and/or mixing chambers in individual modules in orderto drive the flow of one or more reagents through a system. Differentcombinations of pump types can also be realized within a system. Forexample, a block syringe pump can drive targets through the system as awhole, and solid chemical propellant pumps or DEP pumps can beintegrated into individual modules of a system or into individualreagent chambers or reagent loops. Examples of pumps that can be used inthe system include syringes, automated syringe pumps, dielectric pumps,solid chemical propellant pumps, and others known in the art. Differentpump configurations can be realized according to the present disclosure,and one skilled in the art could select a pump configuration dependingon the specific needs of the system.

In certain embodiments, the system can also include a controllerconfigured to control fluid control devices (such as valves and/orpumps) to direct or initiate movement of fluids throughout the system.The controller, for example, can also be configured to open or close oneor more valves to enable transport of the bead complex from the reactionchamber to the analysis region. The system and/or the controller can beelectrically coupled to a computing device as shown in FIG. 23, and thesystem can be configured so that electronic data can be passed to andfrom the system, a controller, and/or a computing device. Configurationof electronic devices and circuits is routine in the art and one skilledin the art would be able to configure the device according to thedesired application.

Computing devices as described herein can execute software that containsinstructions for pump operation, valve operation, controller operation,and can process data from sensors, such as optical sensors which canmonitor and generate real-time data about the environment within thesystem. The system can utilize one or more electronic feedback loopswith sensors coupled to a controller and/or a computing device to assistin automated operation of the system. In certain embodiments, acontroller can be a computing device.

Another embodiment of the present disclosure is a kit that can include amicrobead with a capture molecule coupled thereto and a nanoparticlehaving a probe molecule coupled thereto via a label. The capturemolecule and probe molecule can be configured to be coupled together viaa biological target to form a biological molecule bead complex.

The biological target can be a nucleic acid (e.g., DNA or RNA) molecule.The label can be a spectroscopic dye. The biological molecule beadcomplex can be of a size to fit within a focal point of a laser beamconfigured to analyze the bead complex. The microbead can be at leasttwo orders of magnitude larger than the nanoparticles. The microbead andnanoparticles can be within corresponding collections of multiplemicrobeads and multiple nanoparticles in respective separate solutionsor a common solution.

The kit can also include at least two microbeads having differentrespective capture molecules coupled thereto and at least twonanoparticles having a different respective probe molecules coupledthereto via different respective labels. The different respectivecapture molecules and different respective probe molecules can beconfigured to be, respectively, coupled together via differentrespective biological targets to form a random array of at least twobiological molecule bead complexes in random locations in a fluid.

The label can be configured to provide a Raman spectrum to a portableRaman spectrometer. In some embodiments, the capture molecule is anucleic acid (e.g., DNA or RNA) molecule configured to bind to thebiowarfare agent or a component thereof. At least one of the capturemolecule and probe molecule can be an aptamer, antibody, or DNAmolecule. The microbeads can have multiple capture molecules coupledthereto, and the multiple capture molecules can be the same. Themicrobead can have multiple capture molecules coupled thereto, and themultiple capture molecules can be different. The nanoparticles can begold nanoparticles and can have one or more silver nanoparticlesattached thereto.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

Certain definitions applicable to this disclosure are included herein.

EXAMPLES

Now having described the embodiments of the disclosure, in general, theexamples describe some additional embodiments. While embodiments of thepresent disclosure are described in connection with the example and thecorresponding text and figures, there is no intent to limit embodimentsof the disclosure to these descriptions. On the contrary, the intent isto cover all alternatives, modifications, and equivalents includedwithin the spirit and scope of embodiments of the present disclosure.

Example 1

According to embodiments of the present disclosure, the operation of abreadboard for a system for detecting a target molecule can involveexposing a biological sample to a series of prepared biochemicaltreatments to extract DNA from the sample. The DNA can then be capturedand labeled via specific chemistries attached to the surface of glassbeads, which serve as transport media. Nanoparticles can form part ofstructures attached to glass beads. The beads can then be transported tobe under a laser beam of a surface-enhanced Raman spectroscopy (SERS)sensor that produces a Raman spectrum that is unique to the Raman labelassociated with a gold nanoparticle probe designed to complement to aspecific biological target, such as a nucleic acid molecule from aspore, bacterium, or virus. This unique SERS spectrum of the labelcorresponds only to the specific DNA detected, which is the basis forunambiguous detection and identification.

In some embodiments, following injection of the sample into a receivingvial, the breadboard lyses the sample chemically by mixing it withpreselected enzymes and/or detergents to disrupt and dissolve cell wallsand release the target DNA into solution. Alternative types of lysingcan also be used, such as mechanical or thermal lysing. The DNA can thenbe collected and washed free of cell debris on silica gel contained in aglass capillary. Additional processing can also be performed, such asfragmentation with restriction enzymes. After lysing, purification stepscan follow, e.g., before or after fragmentation. The DNA can then beremoved from the silica gel and hybridized to the complementary DNAstrands anchored (e.g., hybridized) to the surfaces of glass beads. Thebeads, with the target DNA attached, are then reacted (e.g., hybridized)with additional complementary DNA strands which are bound to organic dyemolecules, or other labels, that are themselves bound to goldnanoparticles to give large SERS signals of the dye when analyzed.Additional enhancement can be achieved by reacting the bead-DNA-goldnanoparticle complex with an enhancing solution, which may contain goldor silver ions, for example. This solution will deposit gold or silverparticles onto the pre-existing gold nanoparticles bound to the probeDNA. The SERS signal from the organic dye can be amplified by a factorof a million, for example, thereby providing excellent sensitivity andextreme specificity of the target DNA toward its complementary DNA,ensuring high selectivity for the target. Therefore, by detecting aspecific organic dye by SERS, a specific biological target is detected.

Embodiments of the present disclosure can be used for a portablepathogen, e.g., biological warfare agent (BWA) sensor for use in thefield, for example. Embodiments enable BWA samples, collected in thefield, to be detected and identified in less than 30 minutes using newbiochemistries, processing treatments, microfluidic devices, anddetection methodologies. The organism-specific deoxyribonucleic acid(DNA), or ribonucleic acid, e.g., in the case of virus detection, can beextracted, processed, and detected with high sensitivity andselectivity. This can be achieved using random array analysis processesand detection using surface-enhanced Raman spectroscopy (SERS),according to embodiments of the present disclosure.

One implementation of embodiment devices is a field-use instrumentdesigned specifically for Ebola detection. Based on testing an automatedbreadboard prototype described hereinafter, an Ebola detection deviceutilizing RNA processing chemistry is expected to be able to perform ananalysis to detect Ebola within about 10 minutes.

The breadboard can be utilized to detect many different pathogens,proteins, and other biological targets. When used to detect a BWAtarget, for example, operation of the breadboard can involve thefollowing example procedures. The BWA sample can be exposed to a seriesof mechanical and biochemical treatments to extract DNA from the target,which is then captured and labeled via specific chemistries attached tothe surface of glass beads, which serve as transport media. The beadsare then transported under the laser beam of a SERS sensor that producesa Raman spectrum that is unique to the BWA label. This unique SERSspectrum of the label corresponds only to the specific DNA detectedwhich is the basis for unambiguous detection and identification.Following injection of a BWA sample into a receiving vial, thebreadboard lyses the sample mechanically to disrupt cell walls andrelease the DNA of the BWA into solution. The DNA is then collected andwashed free of cell debris on silica gel contained in a glass capillary.The DNA is then removed from the silica gel and chemically bound tocomplementary DNA strands anchored to the surfaces of glass beads. Thebeads, with the BWA DNA attached, are then reacted with additionalcomplementary DNA strands, which are bound to organic dye molecules thatare themselves bound to gold nanoparticles to give large SERS signals ofthe dye when analyzed. The SERS signal from the organic dye is amplifiedby a factor of about 1 million, thereby providing excellent sensitivityand extreme specificity of the agent DNA toward its complementary DNAensuring high selectivity for the BWA target. Therefore, by detecting aspecific organic dye by SERS, a specific BWA is detected. The prototypebreadboard referred to above and described further hereinafter has beensuccessfully used to detect Yersinia pestis (YP) (spore analyte),Bacillus anthracis (BA) (bacterial analyte), and Venezuelan equineencephalitis (VEE) (virus analyte), for example.

Example 2

FIG. 1A illustrates bead complexes 100 a and 100 b, which can be used inembodiments of the present disclosure for target DNA detection. The beadcomplex 100 b includes a microbead substrate 102 coupled to a captureDNA molecule 104. A gold nanoparticle 106 is coupled to a probe DNAmolecule 110 via a Raman dye 108. The probe DNA 110 is configured to becoupled to the capture DNA 104 via a target DNA molecule 112. Thedesignations of the molecule 104 as the “capture” molecule and themolecule 110 as the “probe” molecule are arbitrary, as either molecule104 or molecule 110 can be considered to perform “capture” or “probe”functions.

The bead complex 100 a illustrated in FIG. 1A is similar to the beadcomplex 100 b but also illustrates that the microbead 102 can be coupledto multiple capture DNA molecules 104. Furthermore, gold nanoparticles106 can each be coupled to multiple probe DNA molecules 110 via Ramandye 108. FIG. 1B illustrates other possible embodiments of beadcomplexes. Nanoparticle use in detecting DNA and RNA molecules haspreviously been described in part in Y. C. Cao, R. Jin, and C. A.Mirkin, “Nanoparticles with Raman Spectroscopic Fingerprints for DNA andRNA detecting,” Science, 297, 1536 (2002), the entirety of which ishereby incorporated herein by reference; and in U.S. patent applicationSer. No. 10/172,428, filed on Jun. 14, 2002, the entirety of which isalso incorporated herein by reference.

It should be noted that the bead complex 100 b is referred to as a DNAbead complex or microbead complex because the capture DNA 104 and probeDNA 110 are configured to bind to the DNA target; however, the targetcan also be an RNA molecule or a protein, and the target and capturemolecules be configured to bind to RNA or protein respectively, forexample.

The Raman dye 108 can be other forms of labels as understood in the artof spectroscopy, such as a fluorescent dye (Alexa Fluor® 488 forexample). Other bead complexes with other types of labels, such asfluorescence labels, can alternatively be used provided they can beidentified by a spectrometer, thus identifying a probe DNA and targetDNA to which they are attached. The microbead 102 and gold nanoparticles106, with their respective DNA molecules attached, can be broughttogether in a reaction chamber (illustrated hereinafter in conjunctionwith FIG. 10, for example) or an assay mixing chamber to form the beadcomplex 100 b. It should also be noted that the complex 100 a in FIG. 1Ahas multiple capture molecules 104 coupled thereto, and the multiplecapture molecules are the same, or homogenous. This can facilitatereaction with a target DNA in a fluid in a reaction chamber. In otherbead complexes, the multiple capture molecules can be differentsequences (ie heterogeneous) to allow a single bead complex to includedifferent target molecules.

Gold (Au) nanoparticles 106 (e.g., 13 nm in diameter) modified withRaman-dye-labeled alkylthiol-capped oligonucleotide strands can be usedas probes to monitor the presence of specific target DNA strands. Thereare over 100 oligonucleotide strands on each Au nanoparticle to ensurerapid and efficient bonding. The specific dyes chosen as the Ramanlabels exhibit large Raman cross sections.

In some embodiments, the microbead 102 is a substantially sphericalglass microbead, but in other embodiments, other shapes and materialsmay be used.

FIG. 1C shows another embodiment of target hybridization and detectionaccording to the systems, devices, and methods herein. A microbead 102is coupled to one or more capture molecules 104 that can have identicalor different sequences to form a microbead complex. A nanoparticlecomplex is formed by coupling a nanoparticle (silver or gold, forexample) 106 to one or more probe molecules 110 (having identical ordifferent sequences) through a dye 108, such as a Raman dye or otherspectroscopic dye. The nanoparticle complex and the microbead complexthen bind to a target molecule 112, such as a DNA molecule, creating ahybridized microbead complex. This reaction can take place in a reactionchamber or assay mixing module as described herein. The microbeadcomplex can be mixed with silver or a silver solution containing silverions or positively charged silver ions as well as a hydroquinone. Alaser with an emission spectra approximately complementary orcomplementary to the absorption spectra of the dye 108 can then shine onthe microbead complex, creating a SERS emission spectra which can thenbe analyzed.

A “random array” design can be utilized, which is ultimatelyimplementable on a microfluidic chip, whereby flowing glass beadscontaining the sandwich assay shown in FIGS. 1A-1C are passed through ananalysis region, which can be the SERS optical probe area. This randomarray assay uses free floating probes in the assay instead of thestationary DNA strands on a substrate in a more conventional microarray.In this type of assay, batches of beads for different but specifictargets are prepared. Reporter groups are used to identify the bead, thecorresponding target, and whether or not a reaction with the targetnucleic acid sequence has taken place. Incorporating the random arraywith a SERS-based reading system inherently assures a high degree ofmultiplexing by the use of multiple dye labeled nanoparticles and thewell-defined Raman spectral features of the dyes. Useful attributesunderlying the potential of the random array methodology include but arenot limited to: fast hybridization kinetics—fast assay and rapiddetection; simultaneous multiple assays in a single cartridge; SERSreadout with fixed optical configuration; high reliability of field unitwith no moving parts; mechanically robust bead storage in cartridge,small cartridge size by scaling only bead volume to add assays;inexpensive to produce; simple and easy-to-use self-contained assaycartridge.

These attributes ensure that the random-array detection system isexceptionally well-suited for the eventual development of a portableDNA-detection cartridge.

An embodiment of a method of DNA detection utilizing devices asdescribed herein is as follows: after input of the sample (e.g.,aqueous) containing the targets of interest, operation of a breadboardcan be based on established laboratory scale procedures as follows:

Cell lysis by lysis buffer and DNA fragmentation by enzyme

Washing and separating DNA through the silica-gel membrane utilizinginherent silica affinity

Transporting the target DNA to the mixing and capture chamber

Injecting microbeads and nano-particle probes into to the mixing/capturechamber for circular flow mixing driven by the dielectrophoresis-pump(DEP) integrated below the chamber

Incubation and capturing target DNA and nano-particle probes onmicrobeads with the aid of thermal cycling to achieve hybridization ofthe probes.

Washing

Flushing with silver (Ag) enhancement solution

Washing

Collecting the microbeads for SERS spectral analysis

Measuring SERS spectrum to detect and identify biological warfare agentspresent

Useful considerations in the assay array implementation include:

Washing/flushing/cleaning. Efficient washing steps are helpful to ensureremoval of unbound probes to avoid false positive results.

Probe selection. Unique Raman dyes can be selected for each probe sothat detection occurs only in the presence of a specific target DNA.

Cell lysis. Bacteria cells can be lysed by mixing with a lysis solution.

DNA fragmentation. By injecting enzymes, the long ds-DNAs extracted frombacteria are fragmented into shorter pieces (such as less than 200 basepairs), which is useful for the hybridization of Raman-reporterparticles. Similarly, long RNAs extracted from viruses are fragmentedwith RNA specific reduction enzymes, then treated similarly to DNA, asdescribed below.

DNA separation. By filtering through a silica-gel membrane, the DNAfragments are absorbed onto the membrane in a high-salt buffer solution.After washing, the fragmented bacterial DNA are released and collectedfrom the membrane using a low-salt buffer solution.

Hybridization. The chemistry for the hybridization of Raman-reportingparticles with bacterial DNA targets onto the beads is a core of theassay. To achieve high-sensitivity and high-selectivity detection, thischemistry can be optimized via (a) detailed design of locked nucleicacid (LNA) or peptide nucleic acid (PNA), (b) the choice of Ramanreporting dyes, (c) the size of gold nanoparticles for the probes, (d)the size of the random array supporting beads (e) the chemistry tofunctionalize the LNA probes onto these beads, and (f) the hybridizationconditions such as temperature, buffer solution, and hybridization time.

The YP cell lysis and DNA extraction protocol optimization was performedby focusing on using the Qiagen DNeasy® blood and tissue kit. Afterexperimentally determining this kit as the most successful commerciallyavailable kit, work was performed to optimize the DNA yield. In thisprotocol, an aliquot of a known number of cells was isolated to belysed. Cell lysis buffer and Proteinase K was added to the cells, whichwere then resuspended and incubated in a water bath. Followingincubation, RNase A was then added to the lysis solution. An additionallysis buffer was added to the solution to finish the lysing anddigestion reactions. Ethanol was then added to the solution toprecipitate genomic DNA from the solution. The solution was transferredto a silica-membrane spin column, where it was washed with two differentbuffers and eventually eluted from the column. The resulting materialwas then measured by UV-Vis spectrophotometry to determine the DNAyield.

The treatment of Gram-Positive bacteria, like BA, was slightly differentfrom the Gram-Negative procedures for the lysis and DNA purification ofYP. In order to lyse the cell wall of BA, an enzymatic lysis buffercomprising 20 mM Tris-Cl pH 8.0, 2 mM sodium EDTA, 1.2% Triton® X-100and 20 mg/mL lysozyme from chicken egg white was first prepared. Thislysis buffer is sufficient for breaking the cell walls of Gram-Positivebacteria and spores because of the aggressive lysozyme and the detergentcomponents. After an appropriate number of cells (here, ˜1×107) wereharvested, they were centrifuged into a pellet and the supernatant wasdiscarded. The bacterial pellet was then resuspended in 180 μL of thepreviously described enzymatic lysis buffer and then incubated at 37° C.for at least 30 minutes. 25 μl of Proteinase K and 200 μl of Buffer ALwas then added and mixed through vortexing. The reaction tube was thenincubated at 56° C. for 30 minutes, causing lysis of the BA cells andallowing for extraction of the BA DNA. After incubation, 200 μL ofethanol was added to begin DNA precipitation. The reaction was mixedtill homogeneous by vortexing. The mixture was then transferred onto theSilica gel of a Qiagen DNeasy® Mini Spin Column in a 2 mL collectiontube. DNA selectively attached to the membrane, driven by the highconcentration of chaotropic salt. The mixture was then centrifuged at8000 rpm (6,046 g) for 1 minute. The DNA remained adhered to the silicagel and the flow-through is discarded. The column was then placed in anew 2 mL collection tube and 500 μL of buffer AW1 was then added to thecolumn and then centrifuged at 8000 rpm (6,046 g) for 1 minute. Again,the flow-through was discarded and 500 μl of buffer AW2 was added to thecolumn. This was then spun down at 13,000 rpm (16,060 g) for 3 minutesin order to dry the silica gel membrane. Flow-through was againdiscarded and the column was placed in a clean collection tube.

At this point, clean DNA remained attached to the silica gel membrane.It was then eluted with 200 μl of Buffer AE added to the spin column;this buffer has a low salt concentration. After 1 minute of incubationat room temperature the column was spun at 8000 rpm for 1 minute. Theflow-through was then collected and measured by UV-Vis spectrophotometryfor DNA content. This process was repeated several times, and eachrepeat yielded about 50% of BA DNA as determined by UV-Visspectrophotometry.

Example 3

FIGS. 2A and 2B illustrate an in-line capillary heater for thermal celllysis. In the device of FIGS. 2A-2B, heating can be achieved by applying3.0 VDC and 1.0 amps to 24 gauge nichrome wire coiled over a 4 mm OD (2mm ID) quartz capillary. A thermocouple to monitor the liquidtemperature is inserted into the capillary from the left in FIG. 2B.

In order to minimize the analysis time for this application, ways wereexplored to minimize the time required for each processing step. Theenzyme-based cell lysing method can be replaced with a more timeefficient thermal lysing procedure. In this method, lysing is achievedby simply passing the liquid cell suspension sample through a capillaryheated to between 95° C. and 200° C. If successful, the cells shouldlyse and the cellular debris dissolve, releasing the cell's DNA forsubsequent capture and purification. To test this method, a heatedcapillary was built by coiling 24 gauge nichrome wire (60% Ni, 12% Cr)around a quartz capillary as shown in FIGS. 2A-2B. The nichrome wire washeated with a DC power supply providing 3.0 VDC and 1.00 Amps. Athermocouple was inserted into the capillary to monitor the sampletemperature. The capillary was filled with a solution of BA spores at aconcentration of 2×10⁸ cells/mL. After filling the capillary, power wasapplied to the nichrome wire. FIGS. 3A-3C illustrate successfulcompletion of this test, showing successful lysis and dissolution ofcellular debris.

FIGS. 3A-3B show micrographs of BA spore solution before and afterheating the temperature through a temperature profile shown in FIG. 3Cwith the device of FIGS. 2A and 2B and with maximum temperature of 195°C. Efficient spore lysis of greater than 95% is indicated by nearabsence of spores remaining after heating sample, as seen in FIG. 3B.The capillary in FIGS. 2A-2B containing the BA spore solution was heatedwith the temperature profile shown in FIG. 3C. These results show thatefficient cell lysing can be performed on a microfluidic platform evenfor cells and material that is resistant to traditional lysis methods,such as difficult-to-lyse BA spores.

Example 4

Initially, experiments were conducted on YP detection using syntheticdouble-stranded DNA (ds-DNA). Results demonstrated the successful SERSdetection of YP at a ds-DNA concentration of 0.1 nM through the randomarray detection format. In this work, an alkylthiol-capped, Cy3-labelledoligonucleotide probe (for binding onto gold nanoparticles), analkylthiol-labeled oligonucleotide probe (for binding onto silicaparticles), a 44-mer double strand DNA target as a positive control:5′-GTG AGA GTA GGA TCA TAT ACC GTT AGA TGC TGC TGG CGT TAT G-3′ (SEQ IDNo. 1) (the sequence is determined in accordance with the informationfrom M. Guelta: Y. Pestis genes GenBank: X61996), and a 44-mer negativecontrol: 5′-GTC ACT CTA GGA TCA TAT ACC GTT ACT TCG ACG TGG CGA TAT G-3′(SEQ ID No. 2) were designed. A method for synthesizing 13-nm diametergold nanoparticles has also been established.

Gold nanoparticles functionalized with Cy3-labelled oligonucleotideswere prepared as SERS probes. The resulting nanoparticles were shown tobe stable in phosphate-buffered saline (0.6 M NaCl). UV-Vis spectroscopyshowed that these particles exhibit a characteristic surface plasmonband of monodispersed gold nanoparticles, a result that was furtherconfirmed with transmission electron microscopy. All together, theseresults show that the synthesis of DNA-capped gold nanoparticle probesis very successful. Oligonucleotide-functionalized glass beads (100micron in diameter) were also successfully prepared.

Data have confirmed that the random-array assay can give rise to strongSERS signals in the presence of the 44-mer double-stranded syntheticDNAs (5′-GTG AGA GTA GGA TCA TAT ACC GTT AGA TGC TGC TGG CGT TAT G-3′(SEQ ID No. 1)), but not in the presence of the negative control DNAs(5′-GTC ACT CTA GGA TCA TAT ACC GTT ACT TCG ACG TGG CGA TAT G-3 (SEQ IDNo. 2)), for example. It should be noted that the sequences given abovecan, alternatively, be expressed with a “C” or “C-” marker near themiddle of the sequence as a visual demarcation between the half of thetarget that binds to the probe sequence and the half that binds to thecapture sequence. This visual identifier has been removed, and thesequences given above are shown without demarcation.

The stability of Cy-3-labelled DNA-gold nanoparticle probes in a PBSbuffer has been tested. Results have shown that the samples are stableat 4° C. (˜7 weeks) and stable at room temperature for more than 10days.

Oligonucleotide-functionalized glass beads (30 micron in diameter) as aYP-DNA assay have been successfully prepared. Samples were made fromYP-DNA assay at the DNA-target concentration of 1 nM (synthetic DNA).13-nm gold nanoparticle probes were functionalized with Rhodamine 6G dye(R6G)-capped probe DNA, and 30-micron glass beads were functionalizedwith capture DNA. After incubation of YP-DNA targets, gold-nanoparticleprobes, and functionalized glass-beads in a PBS buffer (0.3 M NaCl) for30 min, the sample was washed with PBS buffer, and then silver coated onthe surface of the glass beads where gold nanoparticles were present dueto the YP-target binding. The resulting beads were further washed withPBS buffer and were used for SERS measurements using microRaman.

Results from initial stability tests conducted showed that Cy-3-labelledDNA-gold nanoparticle probes were stable in a PBS buffer (0.3 M) for 38days at room temperature, while it was found that aggregation of goldnanoparticles occurred in the sample stored at 4° C. for 11 weeks.Nanoparticle aggregation was found to be a reversible process and theaggregates can be re-dispersed in PBS buffer at room temperature. Thispositive result suggests that keeping these nanoparticles at roomtemperature might be a good condition for nanoparticle storage.

Optimizing the YP DNA assay using the 30-micron glass beads continued.The total DNA hybridization time of the assay was 50 minutes. Aftersilver staining, very strong SERS signals from Cy3 labels were obtainedusing a micro-Raman spectrometer. The assay time can be decreasedsignificantly by increasing the salt concentration of PBS buffer.

A new double helix DNA for YP was prepared. Since real samples of YPwill contain double stranded DNA (ds-DNA) and not single strand DNA ashas been used previously, this synthetic ds-DNA will be used in futureexperiments to establish and optimize the best DNA chemistry for thecartridge. Test results have shown that the DNA-gold nanoparticleconjugates for YP detection are stable for four and half months.

New probes were designed for the program analytes BA, and VEE. Thesedesigns include capture DNA to be bound to glass microbeads, target DNA,and probe DNA containing gold nanoparticles and Raman dyes. The DNAprobe strands, capture strands, and target strands for BA and VEE wereobtained from Bio-Synthesis Inc. The sequences for all DNA used in thisprogram are listed hereinafter.

FIG. 4 shows a SERS spectrum of Cy3 detected from a YP assay.

This spectrum was the result of successfully integrating andperforming 1) preparing the surface of 30 μm diameter glass beads 2)binding the YP capture DNA (c-DNA) onto glass beads, 3) deprotecting,purification, and binding of the YP probe DNA to 13 nm diameter goldnanoparticles (p-DNA), 4) preparation of the YP target DNA (t-DNA), 5)mixing of the c-DNA, p-DNA, and t-DNA for 25 minutes, and 6) treatmentof the sandwich structure with silver staining solution. (c-DNA, p-DNA,and t-DNA as used herein can refer to capture molecule DNA, probemolecule DNA, and biological target DNA respectively).

The SERS-bead assay process was tested and optimized. Initially, theprocess was carried out using conventional/manual laboratory processesand equipment (i.e., glass vials, orbital shaker, pipets, etc.). Allassays have been performed using YP-Target-FW (“FW” denotes “forward”)as the target DNA sequence, which is complementary to the YP-capture andYP-probe sequences. Reaction volumes were ˜2 mL. After the benchtopassays were deemed successful (determined when the Cy3 signal could bereliably detected on individual beads), the assay was transferred to thebreadboard described hereinafter in conjunction with FIGS. 16A-B forautomated processing. Strong absorbance at 260 nm due to DNA absorptionindicates recovery of an amount of the YP DNA contained in the YP cellssupplied by the Edgewood Chemical Biological Center (ECBC) that will besufficient for analysis.

Example 5

FIGS. 5A and 5B are graphs illustrating SERS spectra collected forassays of VEE and BA, respectively. Twenty beads were measured followingeach assay, and each collected spectrum is shown. Building on previouswork that successfully demonstrated the assay chemistry for YP in thelaboratory and on the breadboard of FIGS. 16A-B, assay chemistries forthe analytes BA and VEE were implanted. By following the same stepwisechemical processing procedure developed for YP to bind the capture,target, and probe DNAs onto glass beads, strong SERS signals for both BAand VEE were observed. Note that the probe dye for VEE is Cy3.5 and theprobe dye for BA is Cy5. As shown in FIG. 5, very strong SERS signalswere measured on nearly all beads for both assays, indicating that theefficiency of the chemistry is quite good. It is also observed that theSERS spectra from the probe dyes Cy3.5 and Cy5 are sufficientlydifferent in spectral features to allow ready differentiation betweenanalytes using these two dyes as probes. The integration time for theSERS measurements on each bead was only 10 seconds, indicating thatstrong SERS signals can be obtained using short measurement times forthese well designed assay probes.

FIG. 6 shows SERS spectra collected from VEE and BA negative controlassays. Twenty beads were measured following each assay, and eachcollected spectrum is shown. To demonstrate the selectivity of the BAand VEE assays, negative control experiments were conducted wherein theBA assay was run with VEE target DNA and the VEE assay was run with BAtarget DNA. Strong SERS signals would not be expected on any of thebeads. This is, in fact, the observation as shown by the SERS spectracollected from these negative control assays in FIG. 6. All of thespectra in FIG. 6 are weak in intensity and are readily distinguishablefrom the positive spectra in FIGS. 5A and 5B. The low intensity spectralfeatures in the spectra can be attributed to residual probe materialafter washing and not to nonspecific binding of probe DNA. The broadspectral feature in many spectra that is centered at 1400 cm−1 isassociated with the glass beads themselves.

The improved breadboard can be designed to handle and process 10-20times less sample volume, typically approximately 50-200 than thebenchtop procedure. Automated mixing/reacting of the capture beads,target DNA, AuNP-probes (gold nanoparticle probes), Ag-developmentsolution, and Cl⁻ solution (with intermediate washing steps) was thenaccomplished without involvement from the user. The entire currentprocess for YP (un-optimized) is timed to be approximately 30-40minutes. After the beads have been processed, they are pipetted out ofthe breadboard reaction tube, and transferred to a quartz microscopeslide for analysis.

For each assay, the SERS signals of 20 beads are randomly measured.Measurement parameters are constant at 100 mW and 10 s integration timeusing a 50× microscope objective. Most assays were performed using 1-10nM of target DNA. In these cases, only ˜1-5% of the beads produced asufficient Cy3 signal. Assays to determine the capture beads' bindingcapacity were also performed; this was accomplished by first treatingthe capture beads to an excess of target DNA. After thorough washing,the beads are reacted with AuNP-probes and then treated withAg-development solution. Finally, the beads are treated with Cl— bufferto enhance the SERS response. At these high concentrations of target DNA(˜1 μM), the beads produce approximately the same signal as the beadstreated with ˜1 nM target DNA, i.e., only ˜1-5% of the beads produce ameasurable Cy3 signal.

Example 6

FIGS. 7A and 7B show similarity between A) benchtop and B) breadboard YPassays, respectively, using 1 μM YP target. Twenty beads were selectedrandomly and measured for each assay.

SERS Cy3 spectra of 20 randomly selected beads processed on the benchtopand breadboard are shown. The data show that the assay on the breadboardcan be as good as or better than the benchtop assay, demonstrating theeffective automation of the assay. The intensity scale is fixed at amaximum of 30,000 counts for comparison with data presented later inthis document.

FIGS. 8A and 8B shows SERS measurements on 20 randomly selected fullyreacted beads with A) and without B), respectively, the final Cl⁻treatment. By eliminating this final Cr treatment, the percentage ofbeads producing a strong Cy3 SERS signal increased from 1% to 75%. Thisdemonstrates one way that the automated assay protocol can be optimized.It was found that the final Cl⁻ ‘activation’ step actually impeded theSERS response of the beads. By eliminating the use of Cl⁻ treatment, thenumber of beads that produced a strong Cy3 SERS spectrum increased from1% to 75%. It was found that the final Cl⁻ treatment caused the silvercoating to detach from the beads and agglomerate into clumps. Theremoval of the silver coating from a bead drastically reduced the SERSsignal the bead produced.

Example 7

FIGS. 9A and 9B illustrate additional breadboard or system features,namely a microfluidic channel that can be used for SERS measurements(FIG. 9A) and a heated mixing chamber to accommodate cell preparationchemistry (FIG. 9B). To simulate SERS spectra collection from amicrofluidic chip, a channel (70 μm×3 mm) was fabricated using PDMS anda quartz microscope slide as shown in FIG. 9A. Inlet and outlet portsallowed glass beads, prepared with our assay chemistries, to be injectedto fill the channel with a transfer pipette. This filling methodsimulated the filling of a channel on a microfluidic chip. Once thebeads are placed in the channel, the entire assembly was placed on themicroscope stage of a PeakSeeker™ Raman system for measurement. Sincethe beads were stationary in this case, each bead was translated to bepositioned under the Raman laser beam for measurement, allowing for thecollection of the 20 SERS spectra reported above from individual beads.All SERS spectra in FIGS. 7A-8B were collected using this microfluidicchannel assembly. Also, in order to transition the lysing chemistry tothe breadboard, a heated mixing chamber was built, as shown in FIG. 9B.The heated chamber maintained a temperature of 50-95° C. to denature alltarget DNA to single strand and to prevent nonspecific binding by probeDNA. The heating chamber in FIG. 9B can be configured for conventionalthermal cycling according to known protocols and parameters of nucleicacid binding to assay in optimization of the assay. The heating chamberof FIG. 9B can be a thermal cycler.

Example 8 Design of Cell Lysis and DNA Extraction Functionalities ofBreadboard

A strategy has been developed to implement automated cell lysis and DNAextraction on a system, device, cartridge, and/or breadboard unit. Thebasic processing principles are an extension of the microbead-basedassay shown in embodiments in FIGS. 1A-1C and described throughout;therefore, the use has been extended of this successful design paradigm(e.g., computer-controlled syringe pumping, programmable miniatureelectro-fluidic valves, Teflon capillary fluid handling, miniature glassmixing/reaction tubes, etc.) to also include cell lysis using the lysingchemistry, which includes DNA extraction and purification via asilica-gel-filled capillary.

FIG. 10 shows a schematic diagram illustrating an embodiment of ahardware configuration to incorporate example functionalities. Multipleembodiments of the configuration in FIG. 10 can be realized, for exampleon a breadboard or self-contained microfluidic cartridge coupled to acontroller and/or computing device. The additional components do notincrease the foot-print of the current breadboard, so additional largeequipment (e.g., power supplies, syringe pumps, etc.) is not expected tobe needed.

The breadboard of FIG. 10 includes various three-way valves 1092 andtwo-way valves 1093 to control flow of fluid (ie restrict and/or directamong others) between different chambers of the breadboard, such asbetween the lysing chamber 1090 and an assay chamber 1020. The assaychamber 1020 is the part of the reaction chamber 1820 where nanoparticlecomplexes and microbead complexes as described herein (embodiments shownin FIGS. 1A-1C for example) are reacted or mixed with target molecules.After any reactions have occurred, assay fluid containing any microbeadcomplexes as described herein can be allowed to flow through flow path1822, which can include a microfluidic channel, for example, to ananalysis region such as a capillary analysis region describedhereinafter in conjunction with FIG. 14B. In the analysis region, alaser beam, such as that shown in FIG. 11A, for example, can illuminatedthe microbead structures to produce Raman or fluorescence spectra, forexample, and a spectrometer such as that shown in FIGS. 14A-14B, forexample, can analyze the spectra.

In some embodiments, suction and/or pressure lines are used to produceflow between chambers of the device with the assistance of manuallyoperated or automated pumps.

In the embodiment of FIG. 10, the greater density of glass beadsrelative to the density of the solution in the assay chamber 1020 hasthe feature of allowing bead complexes to settle to the bottom of theassay tube and allowing buffer solution to be extracted via a tube 1021.After extraction, another sample or buffer can be injected through atube 1022. Bubbles in the assay chamber 1020 can provide mixing of beadswith buffer solution in the chamber. In other embodiments, separation ofbeads from solution can be accomplished by filtration.

By controlling flow of fluid between the different chambers of FIG. 10,the valves 1092 and 1093 can cause one or more fluids to assist in theformation of bead complexes in the reaction chamber 1820. Specifically,in FIG. 10, an assay chamber 1020 allows nanoparticles structures andmicrobead structures to react with target molecules (i.e. hybridize) toform bead complexes. Furthermore, a system can also include a controller(not shown) to control the fluid control devices (valves in FIG. 10) tocause the fluids to assist in the formation of the bead complexes in thereaction chamber. The controller, for example, can open or close thevalves, as understood by those skilled in the art of fluid control, toenable transport of bead complexes from the assay chamber 1020 of thereaction chamber 1820 through the channel 1822 to an analysis region. Itshould be noted that the lysing chamber 1090 in FIG. 10 can be a thermallysing chamber, as described in conjunction with FIGS. 2A-3C. However,in other embodiments a lysing chamber can be a chemical or enzymaticlysing chamber or configured to perform lysing by other known methods,for example.

Solenoid valves, plastic tubing and connectors from Lee Company(Westbrook, Conn.), laptop from Dell Corporation, software from NationalInstruments, relay boards from National Instruments, vials, syringe pumpfrom Braintree Scientific, Inc. (Braintree, Mass.), glass microbeadsfrom Polysciences, Inc. (Warrington, Pa.), chemicals from variousvendors, DNA from Bio-Synthesis Inc. (Lewisville, Tex.), heaters fromWatlow Electric Manufacturing Company (Blue Bell, Pa.), power supplyfrom Agilent Technologies (Lexington, Mass.), and Qiagen (Germantown,Md.) DNeasy® blood and tissue kit.

The schematic diagram of FIG. 10 has an expanded breadboard includingcell lysing and silica-gel DNA extraction. The right-side portion of thediagram contains the microbead assay portion. Note that the sizes of thecomponents and tubing lengths are not to scale.

Example 9

Optimization of Cell Lysis and DNA Extraction Functionalities ofBreadboard

Construction of the sample lysing subsystem of the breadboard wascompleted. Lysing of sample cells on a breadboard can be achieved usingheat in conjunction with aggressive enzyme reactions since sonicationmay not be commensurate to the eventual goal of a battery poweredanalyzer for field use. DNA extraction and purification on thebreadboard can be based on a silica-gel-filled capillary.

Example 10

SERS Reader System Design, Build, and Test

A PinPointer™, Agiltron's commercial handheld Raman analyzer (Woburn,Mass.), was used for this program, and the performance of this unit wasevaluated using nanoparticle containing DNA probes. For the bulk of theongoing testing in this program, a PeakSeeker Pro™ from Agiltron(Woburn, Mass.) interfaced with a microscope was used. This highlyflexible setup allowed the best optics for the PinPointer™ to beobtained through optimization testing. As samples were standardized withrespect to size and substrate bead material, the optical train wassimultaneously optimized to obtain the best signal-to-noise ratio SERSmeasurements. The best optical system for the PinPointer™, as determinedvia the PeakSeeker Pro™ tests, was achieved through the iterativeimprovement in the optics to maximize the SERS signal intensity andminimize the noise.

FIG. 11A is micrograph of the 30 μm diameter glass microbead sampleprepared. Shown in the micrograph is the position of the 5 μm diameterlaser beam 1160 for the measurement of the SERS spectrum shown in FIG.11B. The oligonucleotide functionalized microbeads contained Rhodamine6G (R6G) as the probe dye and produced the characteristic SERS spectrumin FIG. 11B. As the first step to evaluate the SERS reader sensitivityrequired to obtain a good signal-to-noise ratio spectrum in a reasonabletime, SERS spectra were collected from initial samples, 30 μm diametermicrobead YP-DNA assays. A 1.0 μL drop of the microbead sample wasplaced on a quartz cover slip to produce the micrograph.

The SERS spectra in FIG. 11B were collected immediately. The spectrum inFIG. 11B is evidence that high quality SERS spectra can be obtained withthe probes prepared and collected rapidly. As can be seen in FIG. 11A,the laser beam 1160 was focused to a small spot size so a relatively fewdye molecules were probed. A much larger (˜100 μm diameter) laser beamcan be used in a handheld SERS reader to sample a much larger number ofprobes. Thus, in some embodiments, a bead complex can fit within a focalpoint or focal region of a laser beam that analyzes the bead complex inconjunction with a spectrometer. The size of the laser orcross-sectional area and/or diameter can be adjusted by a user tooptimize assay sensitivity or to optimize the laser for a particularmicrobead complex. In some embodiments, a microbead or microbead complexcan have a longest dimension (such as a diameter) smaller than a largestcross-sectional dimension of a laser beam. In some embodiments, thelongest dimension of a microbead or microbead complex can be larger thanthe largest dimension of a cross-sectional dimension of a laser beam.Despite the small sampling optical volume, the spectrum in FIG. 11B wascollected using a 10 second integration time and averaging 10 spectrafor a total measurement time of 100 seconds. Using a larger laser beamcan reduce the measurement time to no more than 30 seconds. This resultis of very high significance as it demonstrates that with a programdesigned and synthesized DNA assay, SERS spectra can be generated withsufficient intensity and spectral content for rapid organic analytedetection and identification.

It should also be noted that gold nanoparticles or other nanoparticlesof other metals can be, for example, at least two orders of magnitudesmaller than bead complexes, such as bead complexes 1100 in FIG. 11A.Thus, nanoparticles are not visible in FIG. 11A, and it should also beunderstood that the bead complexes in FIGS. 1A and 1B are not to scale;namely, the microbead 102 can be at least two orders of magnitude largerthan the gold nanoparticles 106.

In many embodiments, the microbeads are between 1 μm and 100 μm, forexample. More preferably, diameters are in a range of between 30 μm and50 μm. In some embodiments, this narrower range allows microbeads to belarge enough for greater visibility during testing or setup, while beingsmall enough to be more easily transported through flow paths.Furthermore, in embodiments in which filtration is done to separatemicrobeads from solution, for example, it can be easier to filtermicrobeads that are larger than about 10 microns in diameter.

FIG. 12 shows a SERS spectrum of a second glass microbead assay samplecontaining Cy3 as the probe dye. The characteristic Cy3 SERS spectrumwas produced. This sample comprised 30 μm diameter glass microbeadssupporting the YP-DNA assay. For these assay samples, Cy3 was used asthe Raman dye in the probe DNA instead of R6G as was used in the firstassay sample prepared. As before, a 1.0 μL drop of the microbead samplewas placed on a quartz cover slip and the SERS spectra were collectedimmediately. The spectrum in FIG. 12 required only one second tocollect, providing evidence that high quality SERS spectra will beobtainable with both the R6G and Cy3 probes prepared. This resultdemonstrates successful multiplexing, namely the ability to generatehigh quality SERS spectra from probes with at least two different Ramandyes with sufficient intensity and spectral content for rapid targetdetection and identification.

FIG. 13 shows a SERS spectrum of a 45 μm diameter glass microbead sample(third sample). The oligonucleotide functionalized microbeads of thethird sample contained Cy3 as the probe dye and produced thecharacteristic Cy3 SERS spectrum.

The third sample, of YP-DNA Bead-AuNP-Cy3 complexes, was, prior to SERSmeasurements, with a vial of 0.3 M phosphate buffer saline solutionmixed and allowed to incubate at room temperature. Immediatelyafterwards, a small droplet of liquid containing active SERS-DNA beads(i.e. microbead complexs) was applied to a quartz microscope slide, andthen a thin quartz coverslip was placed on top of the droplet. The beadswere then measured with the Agiltron PeakSeeker™ system equipped a 785nm laser and 50× long working distance objective. The beam spot size wasslightly larger than the diameter of the beads; each bead was measuredfor 10 seconds with 100 mW power. The spectrum shown in FIG. 13demonstrates the well-defined and exceptionally intense Cy3 signal thatwas measured on the beads. The SERS signal was much more intense whenthe sample was mixed with the phosphate buffer just prior to themeasurement. Previous samples were mixed prior to shipment to the SERSanalysis location and yielded SERS signals only about 10% as intense asthe spectrum in FIG. 13. This result is significant as it leads toincorporating mixing the sample with the buffer just after sampleinjection and filtering and demonstrates that by mixing just aftersample injection and filtering, SERS signals can be significantlyincreased

FIG. 14A-14B show an embodiment of an adapter 1463 fabricated, to testPinPointer™ spectrometer unit 1464 sensitivity for collecting SERSspectra from assay beads in a channel. To demonstrate the feasibility ofhandheld microfluidic applications, a PinPointer™ was tested to collectsignals from actual YP assay glass beads in a 1 mm ID glass capillarychannel (analysis region) 1462.

While a surface enhanced Raman spectrometer is shown in FIG. 14A, otherembodiments can include a fluorescence spectrometer or other type ofspectrometer, for example. Fluorescence spectroscopy can also be used toanalyze the complex if a fluorescent label is used instead of a metalnanoparticle coupled with a Raman dye, for example. Furthermore, whilethe analysis region 1462 in FIGS. 14A and 14B is a capillary, in otherembodiments, the analysis region can have other configurations, shapes,and orientations with respect to the spectrometer. It should also benoted that a spectrometer such as the PinPointer™ 1464 can be coupled tothe reaction chamber 1820 in FIG. 10 through a channel 1822 in FIG. 10on a platform to provide a reaction and analysis system that is mobile.

It should be understood that results of a spectroscopic analysis can bereported to a system user, for example, by any known method. Forexample, a positive test for a pathogen can be reported to a user by anaudible alarm or visual LED indicator, for example. Other example meansof indicating results of a spectroscopic analysis include a text on anLCD screen, WiFi or text message signaling, etc.

FIG. 15 shows SERS spectra of the YP assay incorporating the Cy3 dye.SERS detection of random array assays using the PeakSeeker Pro™ benchtopRaman analyzer interfaced to a microscope system was already proven. Inthis test, SERS spectra collected with the PeakSeeker Pro™ andPinPointer™ were compared. The results, shown in FIG. 15, clearly showthat the PinPointer™ is able to 1) produce SERS spectra characteristicof the YP assay incorporating the Cy3 dye, and 2) produce SERS spectrawith sufficient spectral content for differentiation in an assaymultiplexing system. Thus, more expensive or larger SERS detectionsystems are not necessary.

Example 11

System Integration, Evaluation, and Optimization

FIG. 16A shows an embodiment of a system described herein in abreadboard 1600 configuration used for breadboard testing describedherein. The breadboard system 1600 includes a syringe pump 1666 to drivefluid movement, a lysing station 1668, a DNA purification station 1670,and an assay station 1672.

As the buildup of the breadboard system is complete, all of thechemicals, buffers, and biochemicals necessary for operation can beincorporated into the automated operation of the random array assays.

FIG. 16B is another photograph pointing out other features of thebreadboard 1600 configuration shown in FIG. 16A. The subsectionshereinafter describe the operation of certain features of the breadboardof FIGS. 16A-16B.

FIGS. 16A-16B show embodiments of a system as described hereincomprising a syringe pump 1666 on an automated drive block to createfluid movement through the system, a lysing station 1668 (i.e. a celllysing module), a DNA purification station 1670 (i.e. a biologicaltarget purification module), and an assay station 1672 (i.e. an assaymixing module).

The system of FIGS. 16A and 16B is configured to be run via controlsoftware implemented in LabView on a computing device or apparatus 1010(not shown). A syringe pump 1666 is mounted to a drive block 1674configured to drive a syringe 1675. The syringe can be connected to acomputing device or apparatus 1010 (not shown) via an RS-232 ‘phonejack’ terminal (not shown). This RS-232 cord connects to an adapter witha 9 pin connector, which in turn connects to another adapter that endswith a standard USB cable. The USB cable is plugged into the computer.Wires coming from an electronics box 1678 are connected to theappropriate pin inputs on the syringe pump 1666.

The breadboard 1600 includes four National Instruments 8-channel solidstate relays 1679 in the electronics box 1678. As used herein, acontroller can be the electronics box 1678, for example, and can becoupled to a computing device or apparatus such as the one described inapparatus 1010. USB cables from the four relays 1679 are plugged intorespective relay boxes. These cables are plugged into corresponding USBslots on a USB dock, which is plugged into a USB input on the computer(apparatus 1010, not shown). A 20V power supply (not shown) with leads1688 (example of electrical coupling, electrical communication, and/oroperational coupling) provides all the necessary power to the fluidicvalves, optical sensors, and cooling fan.

Setting up a fluidic system as described herein is now describedaccording to the embodiments in FIGS. 16A and 16B. There can be foursample/reagent ‘loops’ (an Ag development loop 1682, AuNP probe loop1683, capture bead loop 1684, and sample target loop 1684, also shownschematically in FIG. 10 and FIG. 27, for example) presented at thefront edge of the breadboard 1600, and each of these is a ˜10 cm longTeflon tube with MINSTAC (threaded) connectors on both ends. One end ofthe sample/reagent loop is connected to a tan-colored 3-way manifoldthat delivers each of the samples/reagents to a central line that leadsto the reaction/assay vial. The other end of the sample/reagent loopsconnects to a 2-way valve located directly below the aforementionedcentral tube line. The four sample/reagent loops can be unscrewed frommanifolds and a 2-way valve to flush out the sample/reagent loops withde-ionized (DI) water in a squirt bottle. Water can be removed fromsample/reagent tubes, and residual water from the threaded connectionports can be soaked up.

Each sample/reagent loop can hold a liquid volume of up to 70 μL so eachloop is filled with ˜70 μL of solution. Although several ways may beused to fill the loop with a predefined volume, one way is to use apipet to withdraw 70 μL into a pipet tip (not shown), and then carefullyhold the pipet tip flush against the tip of the Teflon tubing of thesample/reagent loop. The liquid is slowly injected into the tube, takingcare to avoid letting the liquid drip out where the pipet tip and tubecome together. To avoid dripping, the tip and tube are brought togetherin a firm and flush manner. After the liquid sample has filled the tube,the end of the tubing loop is connected first to the 2-way valve andfinger tightened. Note that gravity tends to drain the liquid out of theloop if the tube is held in a vertical manner. Thus, the tube is heldsubstantially horizontal until the first end of the tubing is connectedto the 2-way valve.

For the fourth loop 1682, which holds the two components of the AgDevelopment step (initiator and enhancer), 30 μL of one component areadded first, and then a pipet is used to inject a small plug of air (˜1cm) immediately behind the first component. Afterward, 30 μL of thesecond component are then injected. The air plug keeps the componentsseparated so that they do not mix/react until injected into the assayvial 1687.

After filling each sample/reagent loop and connecting to the 2-wayvalves and manifolds, the loop tubes are finger tightened to ensure firmconnections. Next, the NO3-buffer vial 1681 is checked for sufficientbuffer (at least a ¼ full). The buffer is replaced if it is more thantwo days old. The waste vial 1686 then emptied if it is more than ¾full. Note that in order to empty the waste vial, the tubes remainconnected to the waste vial's lid. The vial is carefully removed fromthe vial holder prongs, and the vial is twisted to unscrew it from thelid (this prevents breaking of the seals on the tubing connected to thelid).

To set up the syringe pump 1666, the large 60 mL syringe 1675 isdisconnected from a female luer adapter 1676 (which connects to a reliefvalve 1677 and the main tubing line that drives the rest of thebreadboard pneumatically). The syringe 1675 is attached to the syringepump with the syringe withdrawn to about ¾ its total capacity. Thedriving block on the syringe pump is adjusted so as to have itapproximately align with the end of the syringe plunger when attached tothe pump. The clamps that hold the syringe and plunger in place arechecked for being tightly secured and that there is no extra room forthe syringe to move around. After the syringe is set up, the lueradaptor is reattached to the syringe pump. The threaded fitting on theTeflon tubing is checked for being securely screwed into the plasticluer adaptor.

Setting up Assay Vial on Breadboard. The glass assay vial 1687 and thesmall Teflon tubing adaptor (not shown) are cleaned. Sonication in waterremoves most particulates from the vial. The vial is rinsed thoroughlywith DI water and EtOH and blow dried.

The small Teflon tubing adaptor is attached into the central threadedhole on the plastic base of the assay vial holder. The clean glass assayvial is slid onto the short length of Teflon tubing sticking upwards outof the plastic base (not shown). The rubber cap is attached with severalsmall holes onto the top end of the glass vial. There is a square pieceof Teflon with a large central hole that can be attached to the fourthreaded rod posts surrounding the assay vial. The assay vial isdesigned to fit into the central hole. This square block simply helps tohold up the glass vial in a vertical manner. There are two tubes thatneed to be inserted through the rubber cap and into the vial: thesample/reagent injection tube and the waste/supernatant removal tube.The sample/reagent injection tube is inserted ˜½ to ⅔ of the way intovial from the top (<1 cm or so from the bottom of the assay vial). Thewaste/supernatant removal tube is inserted almost to the very bottom ofthe assay vial (where the taper becomes very narrow). About 1-2 mm areleft between the tip of this tube and the bottom surface of the assayvial.

Running the Assay. The Lab VIEW breadboard control program is started ona computing device or apparatus 1010 (not shown). During each step ofthe assay, the syringe pump is normally either ‘infusing’ or‘withdrawing’, and corresponding LEDs turn on/off in the electronicsbox. The movement of liquids in the system can be observed by eye ateach step to ensure that the software and/or hardware is operatingproperly. Sensors, such as optical sensors, can also be implemented inthe system to create feedback loops which electrical components of thesystem (such as the controller and/or computing device) can use foractive, real-time, monitoring of system and/or assay status. An exampleof sensor implementation is shown in FIG. 10.

The main observations are as follows. During the sample or reagentinjection steps, the corresponding liquids should be flushed out of thesample/reagent loops and into the assay vial. A small pellet of beadsshould be observed that quickly settles to the bottom of the assay vial,and these beads should always be present throughout the assay. However,the pellets should be mostly dispersed throughout the liquid during themixing steps. Bubbles are injected from the bottom of the assay vialduring the mixing step, and only during this step. NO₃-buffer iswithdrawn from the buffer vial during the ‘withdraw buffer into bufferloop’ step, fills the buffer loop, and stops filling the tubing when thefront end of the buffer plug reaches an optical sensor. Additionally,the buffer in the buffer loop is emptied into the assay vial. Thesupernatant liquid is withdrawn from the assay vial after each mixing orincubation process. After the initial mixing/incubation steps (with AuNPprobes), and after the excess probe solution (pink colored) has beenremoved and the beads are washed with NO₃-buffer, the bead pellet shouldhave a definitive pink color if the beads/targets/probes are allcomplementary. If the beads are pink as described above, then theyshould begin to turn a brownish color within a couple of minutes ofinjecting/mixing the Ag Development solution. The drive block on thesyringe pump should not reach the back stopper of the pump, or it willstall.

Example 12

Microfluidic Channel Demonstration Device

FIG. 17A shows 6 inch transparency mask illustrating single- andsheath-channel designs (1774 and 1776, respectively) for microfluidicchannels that can be used to transport and/or analyze microbeadcomplexes as described herein. The widths of the channels (in microns)are indicated for each next to each pattern. FIG. 17B provides amagnified view of a sheath channel pattern with 60 μm wide channels.FIG. 17B shows the hydrodynamic focusing observed with green foodcoloring being pumped into the outer (sheath) channels 1778, and redfood coloring pumped into the center channel. The picture was acquiredwith a 5× objective.

Microfluidic channels like those of FIGS. 17A-17B were fabricated usingtraditional polydimethylsiloxane (PDMS) molding techniques. The mold wasfabricated onto a 6 inch diameter silicon wafer. Positive-tonephotoresist was spin coated onto the wafer, and a transparency maskshown in FIG. 17A was used to selectively expose the photoresist film toUV-light. The transparency mask was designed to have numerous channelpatterns/dimensions. Deep reactive ion etching (DRIE) was then used toanisotropically etch the patterned wafer, so that only the exposed waferregions were etched. The wafer was etched to a depth of 57 um. PDMS wasthen prepared and poured over the patterned/etched wafer; the resultingPDMS film thickness was approximately 1 mm. The PDMS-coated wafer wasthen cured at ˜100° C. for at least 5 minutes. After curing, the PDMSfilm was peeled off the molding wafer, and then individual channelpatterns were cut from the film. The PDMS pieces were then treated withthe electrical discharge from a tesla coil to chemically activate thesurface, which was then applied to a Si wafer or glass microscope slideand allowed to bond for several hours.

Through-holes were then punched into the PDMS over the inlet/outletreservoirs, and 1 mm outer-diameter Teflon tubing was inserted intothese ports. Red and green food coloring were used to determine the flowpattern of the channels, as illustrated in FIG. 17B. The food coloringwas loaded into syringes, which were attached to a syringe pump (notshown); the syringe pump was set to a pump rate of 20 μL/min. FIG. 17Billustrates the level of flow control achieved with this system.

FIG. 18 is a block diagram illustrating a system 1800 for detecting atarget biological molecule, such as the target DNA molecule 112illustrated in FIGS. 1A and 1B. The system 1800 includes a reactionchamber 1820 that enables formation of a biological molecule beadcomplex, such as the bead complexes 100 a and 100 b shown in FIG. 1A. Achannel 1822 is configured to transport the bead complex from thereaction chamber 1820 to an analysis region 1824, and a spectrometer1826 is configured to analyze the bead complex at the analysis region1824.

FIG. 19 illustrates an embodiment method 1900 of detecting a biologicaltarget molecule. At 1940, a biological bead complex is formed in areaction chamber, such as the reaction chamber 1820 illustrated in FIG.10 and FIG. 18. At 1942, the bead complexes transported from thereaction chamber to an analysis region through a channel, such as amicrofluidic channel like the channels 1778 and 1780 illustrated in FIG.17B. At 1944, the bead complex is spectroscopically analyzed at theanalysis region by a device such as a Raman spectrometer. Spectroscopicanalysis can include, for example, surface enhanced Raman spectroscopyor surface enhanced resonance Raman spectroscopy. A stimulation signal(e.g., laser beam) that causes the bead complex to react to produce aRaman spectrum can be part of the spectrometer 1826. In someembodiments, the spectrometer 1826 is another type of spectrometer, suchas a fluorescence spectrometer.

FIG. 20 is a schematic illustration of an embodiment of a microbead kit2000. The kit 2000 can include one or more microbead 2050 to which oneor more capture molecules 2052 are coupled. The kit 2000 also caninclude one or more nanoparticles 2052 coupled to one or more probemolecules 2058 via a label 2056. The nanoparticle[s] 2054 can be a goldparticle, for example, such as the gold nanoparticles 106 in FIG. 1B.The nanoparticles 2054 can also be another type of metal (e.g., noblemetals) with a core shell structure that can produce a proper plasmonicresponse for SERS with a given label, such as silver, copper, aluminum,platinum, nickel, etc., or combinations thereof. Furthermore thenanoparticles 2054 can have other nanoparticles attached thereto. Forexample, the gold nanoparticles 106 in FIG. 1B can have one or moresilver nanoparticles attached thereto in order to shift the resonancefrequency of the gold nanoparticles 106 to match the Raman dye 108 toproduce a stronger surface enhanced resonance Raman spectroscopy signal.The probe molecule 2058 and capture molecule 2052 are configured to becoupled together via a biological target to form a biological moleculebead complex. The biological target can be, for example, a target DNAmolecule such as the target DNA molecule 112 in FIGS. 1A-B. However, thebiological target (not shown in FIG. 20) can also be a protein or an RNAmolecule (e.g., from a virus), for example. Homologous or heterogenouscapture molecules and homologous or heterogenous probe molecules can beused.

FIG. 21 illustrates that different microbeads can have differentrespective capture molecules coupled thereto (heterogenous capturemolecules), and different nanoparticles can have different respectiveprobe molecules (heterogenous probe molecules) coupled thereto by thesame or different respective labels. The different respective capturemolecules and different respective probe molecules can be designed to becoupled together, respectively, via different respective biologicaltargets to form a random array of at least two biological molecule beadcomplexes in random locations in a fluid. The random array 2100 includesa fluid 2182 having two microbeads 102 in different, random locations inthe fluid. Furthermore, there are two gold nanoparticles 106 in thefluid 2182, which include different probe molecules 110 and 110′,respectively, coupled to the gold nanoparticles 106 via different Ramandyes 108 and 108′, respectively. The probe molecule 110 and capturemolecule 104 are configured to be coupled together via a target molecule112. A target molecule 112′ is also contained within the fluid 2182, andthe probe molecule 110′ and capture molecule 104′ are configured to becoupled together via the target molecule 112′. In this way, differentbiological agents, such as the different target molecules 112 and 112′can be part of a random array. Furthermore, when the beads andnanoparticles are coupled together via reaction with the targetmolecules, the respective bead structures formed thereby can flow in thefluid 2182 through a flow path such as the flow path 1822 in FIG. 10 toan analysis region such as the capillary analysis region 1462 in FIGS.14A and 14B be to be spectroscopically analyzed simultaneously. Aspectrometer such as the Pinpointer™ 1464 shown in FIG. 14A candistinguish the bead structures from each other via respective Ramanspectra produced by the different Raman dyes 108 and 108′. Thus, a kitthat includes the respective beads 102 and respective nanoparticles 106can be used to form different bead complexes that can be analyzedsimultaneously in a random array.

It should be noted that other embodiments of kit 2000 can includemicrobead 2050 and the nanoparticle 2054 as part of correspondingcollections of multiple microbeads and multiple nanoparticles. Thecollections of multiple microbeads and multiple nanoparticles can belocated in a fluid solution 2182 (described hereinafter in conjunctionwith FIG. 21) or in separate solutions (not shown). When reactions withpotential biological agents are desired, respective solutions can bebrought together with target DNA molecules, for example, in a reactionchamber such as the chamber 1820 in FIG. 10.

As one alternative to the bead structures illustrated in FIGS. 1A-1B andFIG. 21, a probe molecule can be directly immobilized onto a surface ofa gold nanoparticle independently of the Raman label. For example, thiscan be achieved by using a probe sequence with a thiol-modified terminalgroup, which will covalently immobilize the probe molecule to thenanoparticle surface. The Raman label (e.g., Cy3) can then beco-immoblized with the probe molecule (either sequentially or inparallel) onto the surface. This can allow the probe sequence and labelto be immobilized onto the nanoparticle in various yet controllableratios, as opposed to the fixed 1:1 ratio achieved when the probesequence is covalently bonded to the Raman dye, and immobilizedtogether. One advantage of including a plurality of labels for eachprobe molecule is a corresponding increase in Raman signal.

Example 13

FIG. 22 is a schematic illustration of a design for a random array assaysolid state cartridge implementation of an embodiment system 2200 orsystems otherwise described herein. The operation is describedhereinafter in reference to a bacterial input sample. However, theembodiment system 2220 can also be configured to analyze spores,viruses, and other pathogens or biomarkers. After input of the aqueoussample containing the target bacterium of interest, operation of thecartridge (i.e. method of use) is based on established laboratory scaleprocedures as follows:

1. BWA filtering at a filter 2261 to concentrate the target bacterium.

2. Cell lysis by lysis buffer and DNA fragmentation by enzyme, heat, ormechanical means at 2262.

3. Washing and separating DNA through the silica-gel membrane utilizinginherent silica affinity at 2264.

4. Transporting the target DNA at 2266 to the mixing and capture chamber

5. Injecting microbeads and nano-particle probes into to themixing/capture chamber at 2268 for circular flow mixing.

6. Incubation and capturing target DNA and nano-particle probes onmicrobeads with the aid of thermal cycling, also at 2268.

7. Washing using buffer solution 2270.

8. Flushing with an Ag enhancement solution 2272.

9. Washing again using buffer solution 2270.

10. Aligning the microbeads by fluidic focusing for SERS spectralanalysis at 2274.

11. Analyzing a SERS spectrum to detect and identify biological warfareagents present.

The cartridge is non-mechanical—it incorporates only solid statemicrofluidic (e.g. configured for nanoliters, microliters, picoliters,femtoliters) components—and it is powered by the reader unit (notshown). A chemical lysis process has been selected over a processinvolving sonication, but in other embodiments, other processes such asthermal or mechanical lysing may be successfully employed.

The performance of an example commercially available mechanical celllysing device for integration into a breadboard or microfluidic analysissystem was investigated. Specifically, the efficiency of the lysingdevice was determined for Bacillus subtilis (BS) spores strain 168 (ATCC23857) acquired from American Type Culture Collection (ATCC). The devicetested was the commercially available OmniLyse® bead blender fromClaremont BioSolutions (Upland, Calif.).

Cell lysing efficiency was determined by measuring the BS intact cellsuspension concentration and comparing the result to the amount of DNArecovered following extraction and purification. The BS cell suspensionwas measured following the procedure of measuring the cell suspensionturbidity using UV-Vis spectroscopy. The result was an absorbancemeasured at 290 nm of A290=0.255. The A290 value can be converted toconcentration in g/L using Ccell=(A290/3.84 L/g)D where D is thesolution dilution factor, 100 in this case. The result is 6.64 g/L. Toconvert this result to cells/mL, the wet density (1.223 g/cm³) andvolume (0.160 μm³) of BS cells are used to obtain 3.4×10¹⁰ cells/mL.

The amount of DNA recovered from the sample was obtained by measuringthe absorbance of an aqueous solution at 260 nm to give A260=0.60. Themeasured absorbance value was converted to a concentration using[DNA]=A260D (50 μg/mL), where D is the dilution factor (1 in this case)to give 30 μg/mL. Since the number of base pairs of BS double strand DNA(dsDNA) is known (4,214,810) and the conversion factor of base pairs tomolecular weight (MW) formula of MWdsDNA=4,214,810×607.4+157.9 is wellestablished, the BS molecular weight is readily calculated to be2.56×109 g/mole. The number of molecules of BS dsDNA measured was thencalculated: NDNA=(30×10⁻⁶ g/mL)×(6.023×10²³/mole)/2.56×10⁹g/mole)=7.06×10⁹ mL⁻¹.

The efficiency of the DNA extraction process, which includes celllysing, is the ratio of molecules of DNA per mL to the number of cellsper mL, 7.06×10⁹ mL-1/3.4×10¹⁰ mL⁻¹=0.21. It is also noteworthy that thelysing step was performed in only 3 minutes.

Key considerations in the assay array implementation include:

Sample pre-concentration. The cartridge will include a component toconcentrate organisms from liquid samples. This can be built using a setof filters with different pore sizes: the larger pore sized filters(e.g., 20 μm) can be used to filter out the large sized impurities andsmaller pore sized filters (e.g., 0.22 μm) can be used to concentratethe target organisms. This step can be performed by or implemented insample isolation modules as described herein.

Cell lysis. The bacteria cells are lysed by mixing with the lysissolution that is injected from its storage by the integrated solidchemical propellant pump. Alternatively, cells can be lysed by heatingor mechanical blending or a combination of all. This step can beperformed by or implemented in cell lysis modules as described herein.

DNA fragmentation. By injecting enzymes, the long ds-DNAs extracted frombacteria are fragmented into shorter pieces (such as less than 200 basepairs), which is important for the hybridization of Raman-reporterparticles. This step can be performed by or implemented in cell lysismodules as described herein, or in other modules, wherein for examplerestriction enzymes can be added to the module for DNA fragmentation.

DNA separation. By filtering through a silica-gel membrane, the DNAfragments are absorbed onto the membrane in a high-salt buffer solution.After washing, the fragmented bacterial DNA will be released andcollected from the membrane using a low-salt buffer solution. This stepcan be performed by or implemented in biological target isolationmodules as described herein.

Microbead complex formation. Isolated DNA fragments are hybridized tomicrobeads containing capture molecules and nanoparticles containingprobe molecules coupled to the nanoparticles with a Raman dye. This stepcan be accomplished with the aid of heat, thermal cycling, mixing,bubbling, or other methods known in the art. Specific configuration ofthe microbead complexes and nanoparticle complexes can be realized by anend user according to the target molecule and desired analysis method.This step can be performed by or implemented in assay mixing modules asdescribed herein, and the module can be configured according to thedescription herein. Movable separation walls (2267 in FIG. 22) can beincorporated into the device to aid in selective compartmentalizationfor microbead complex formation.

Transport and analysis of the microbead complexes. Transport of themicrobead complexes (which can be a microbead coupled to a capturemolecule hybridized to a target molecule, wherein the target molecule isalso hybridized to a nanoparticles complex which comprises ananoparticle coupled to a probe molecule via a dye) from an assay mixingmodule or the system otherwise to an analysis region where analysis cantake place can be accomplished with a microfluidic channel as describedherein.

Example 14—Examples of Procedures for Random Array DNA Analysis Example14.1: Bench Top Lysis/Assay Procedure

Lysis: Gram-Negative Bacteria (Yersinia pestis (VP), for Example)

Collect cells (maximum 2×10⁹ cells) in a microcentrifuge tube (Sampleprovided by ECBC) by centrifuging for 10 min at 5000 g (7500 rpm).Discard supernatant.

Resuspend pellet in 180 μL Buffer ATL (Qiagen DNeasy® Blood and TissueKit).

Add 20 μL Proteinase K (Qiagen, DNeasy®, 600 mAU/mL solution). Mixthoroughly by vortexing, and incubate at 56° C. for 1 hour (can be lysedovernight if necessary). Vortex occasionally during incubation todisperse the sample. Vortex a maximum of 15 pulses.Add 4 μL RNase A (100 mg/ml), mix by vortexing (maximum, 15 pulses) andincubate for 2 min at room temperature.Vortex a maximum of 15 pulses. Add 200 μL Buffer AL (Qiagen DNeasy®Blood and Tissue Kit) to the sample, and mix thoroughly by vortexing(maximum, 15 pulses).Then add 200 μL ethanol (96-100% purity), and mix again thoroughly byvortexing (maximum, 15 pulses).Pipet the mixture from the previous step (including any precipitate)into the silica gel column (DNeasy® Mini spin column) placed in a 2 mLcollection tube. Centrifuge at 6000 g (8000 rpm) for 1 min. Discardflow-through and collection tube.Place the silica gel spin column in a new 2 mL collection tube, add 500μL Buffer AW1 (Qiagen DNeasy® Blood and Tissue Kit) and centrifuge for 1min at 6000 g (8000 rpm). Discard flow-through and collection tube.Place the silica gel spin column in a new 2 mL collection tube, add 500μL Buffer AW2 (Qiagen DNeasy® Blood and Tissue Kit) and centrifuge for 3min at 20,000 g (14,000 rpm) to dry the silica membrane. Discardflow-through and collection tube.Place silica gel spin column in a clean 2 mL microcentrifuge tube andpipet 200 μL Buffer AE (Qiagen DNeasy® Blood and Tissue Kit) directlyonto the silica membrane. Incubate at room temperature for 1 min, andthen centrifuge for 1 min at 6000×g (8000 rpm) to elute.Collect flow-through for DNA SERS Assay.Measure dilution of sample with UV-Vis for DNA quantification.Lysis: Gram-Positive Bacteria (BA, or Bacillus Anthracis for Example)Enzymatic lysis buffer:20 mM Tris-Cl, pH 8.02 mM sodium EDTA1.2% Triton® X-10020 mg/mL lysozyme (Lysozyme from chicken egg white—Sigma Aldrich)*Do not add lysozyme until immediately before use.Collect cells (maximum 2×10⁹ cells) in a microcentrifuge tube (Sampleprovided by ECBC) by centrifuging for 10 min at 5000×g (7500 rpm).Discard supernatant.Resuspend bacterial pellet in 180 μL enzymatic lysis buffer (recipeabove). (Immediately before use, add lysozyme to the lysis buffer).Incubate for at least 30 min at 37° C.Add 25 μL Proteinase K and 200 μL Buffer AL (Qiagen DNeasy® Blood andTissue Kit). Mix by vortexing (on maximum, 15 pulses).Incubate at 56° C. for at least 30 min.Add 200 μL ethanol (96-100% purity) to the sample and mix thoroughly byvortexing (maximum 15 pulses).Pipet the mixture into the silica gel column (DNeasy® Mini spin column)placed in a 2 mL collection tube. Centrifuge at 6000×g (8000 rpm) for 1min. Discard flow-through and collection tube.Place the silica gel spin column in a new 2 mL collection tube, add 500μL Buffer AW1 (Qiagen DNeasy® Blood and Tissue Kit) and centrifuge for 1min at 6000×g (8000 rpm). Discard flow-through and collection tube.Place the silica gel spin column in a new 2 mL collection tube, add 500μL Buffer AW2 (Qiagen DNeasy® Blood and Tissue Kit) and centrifuge for 3min at 20,000×g (14,000 rpm) to dry the silica membrane. Discardflow-through and collection tube.Place the silica gel spin column in a clean 2 mL microcentrifuge tubeand pipet 200 μL Buffer AE (Qiagen DNeasy® Blood and Tissue Kit)directly onto the DNeasy® membrane. Incubate at room temperature for 1min, and then centrifuge for 1 min at 6000×g (8000 rpm) to elute.Collect flow-through for DNA SERS Assay.Measure dilution of sample with UV-Vis for DNA quantification.Preparation of the DNA-AuNP Probe Conjugates and Capture DNAFunctionalized Glass Beads:DNA-AuNP Probe Conjugates (for Small 4 mL Reaction):Cleave/De-Protect the Probe Oligonucleotide:Equilibrate the NAP10 column with 5 mL of 10 mM PO₄ ³⁻ pH7.4 buffer andallow to pass through column; repeat 5-7 times.Add 160 μL of 15.6 mM PO₄ ³⁻ pH7.4 buffer to Probe stock (e.g. 90 μL of100 μM stock; 9 nmole) to bring total solution volume to 250 μL and PO₄³⁻ concentration to 10 mM.Add >10× molar equivalent of tris(2-carboxyethyl)phosphine (TCEP) (e.g.2 μL of 100 mM; 200 nmole) to the Probe oligonucleotide solution. Allowreaction to proceed ˜10 minutes at room temperature with occasionalmixing.Add the 250 μL of Probe oligonucleotide to the NAP10 column and allowthe blue oligonucleotide solution to pass into the column. After soakinginto column, add ˜2 mL of 10 mM PO₄ ³⁻ pH 7.4 buffer to the column andcarefully observe the movement of the colored Probe band towards thebottom of column.Count the clear drops eluting from the column; when the drops becomeblue in color, collect eluted oligonucleotide into a 1.5 mL tube untilthe drops become clear again.Collect droplets #25-41; finally yield ˜0.6 mL.Mix AuNP (13±1 nm Diameter) with De-Protected Probe Oligonucleotide inGlass Vial:Start with 0.55 mL de-protected Probe oligonucleotide (<9 nmole) in 10mM PO₄ ³⁻ pH7.4 buffer (from step e above)Add 1.118 mL of 16.1 nM AuNP (0.018 nmole). This yields AuNP: Probe tobe 1:500 (mol:mol). Also add 1.218 mL of 10 mM and 13.5 μL 1 M PO₄ ³⁻pH7.4 buffer to bring final volume to 3 mL and buffer concentration to10 mM.Mix for 8 hours on orbital shakerAdd 104 μL of 1.5 M NaCl to yield 0.05 M Cl⁻; mix for another 8 hoursRepeat step d until [Cl⁻]=0.15 M; then add 521 μL to yield 0.3 M; mixfor another 8 hours.Centrifuge AuNP-Probes @ 13 k rpm for 20 min; discard supernatant andadd 1 mL of 0.3 M Cl⁻ 1/10 mM PO₄ ³⁻ pH 7.4, and mixRepeat step f two more times, but re-suspend in RNAse-free water;AuNP-Probe should be finally re-suspended in 100 μL RNAse-free water;store in refrigerator at >6° C.Capture DNA-Functionalized Glass BeadsFunctionalize Glass Beads with SMPBMeasure 0.35 g of glass beadsPlace in 25 mL glass beaker, and add piranha solution (15 mL of H₂SO₄and 5 mL of H₂O₂); heat on hot plate at ˜130° C. for 20-30 minutesAllow to cool, pour out piranha solution and rinse 5-10 times with 18MΩ·DI waterImmerse glass beads in 5 mL of aqueous solution in polystyrene dish with1% APS and 0.01% glacial acetic acid (by volume)Rotate sample on an orbital shaker for 90 min at room tempWash the silane-treated modified glass beads 3× with DI waterTransfer glass beads to small glass beaker/vial and dry with N₂; thenbake in oven at 130° C. for 10 min.Promptly treat beads with 5 mL of 1 mM SMPB in 4:1 EtOH:DMSO solution inglass container. Allow the coupling reaction to proceed for 3 h.Rinse the beads 4× with EtOH and dry under N₂.De-Protect/Reduce Capture Oligonucleotide.Equilibrate NAP10 column with 1×PBS and allow to elute through; repeat5-7 times.Thaw 190 μL aliquot of stock (100 μM) Capture oligonucleotide sequenceand add 60 μL of 4.16×PBS buffer (diluted from 10× stock) to yield 250μL of 140 mM Cl⁻/10 mM PO₄ ³⁻. Add >10× molar equivalents of TCEP (e.g.4 μL of 100 μM stock).Allow to incubate at room temperature for 10-11 minutes with occasionalmixing.Add the 250 μL of Capture oligonucleotide to the NAP10 column and allowthe oligonucleotide solution to pass into the column. After soaking intocolumn, add ˜2 mL of 1×PBS bufferCount the clear drops eluting from the column.Couple the De-Protected Thiol Capture Oligonucleotide to theSMPB-Modified Glass BeadAdd 2 mL of 1×PBS to each dish of dried beadsFor each Capture oligonucleotide, add 100, 550, and 150 μL from Tube 1,2, and 3, respectively, to the functionalized beadsAllow the reaction to proceed overnight at room temperature.Rinse the beads 3× with 1×PBS

Example 14.2: SERS Bead DNA Assay Procedure

Hybridization Buffer=0.6 M NaCl, 0.01 M PO₄ ³⁻, pH 7.4

NO₃ ⁻ Buffer=0.6 M NaNO₃, 0.01 M PO₄ ³⁻, pH 7.4

Assay (for Low Target Concentration)

Mix Capture Beads and Target and AuNP Probes in glass vial

1.980 mL of Hyb. Buffer

4 μL Capture Beads

21 μL of ssDNA synthetic Target

18.4 μL of AuNP probe

Incubate on shaker for 20 minutes (for heating see below)

Pipet and discard liquid

Wash

Add 3 mL of NO₃ ⁻ Buffer, briefly swirl vial

Allow beads to settle, then pipet off wash liquid

Repeat steps 8a and 8b two more times

Ag development

Add 6 drops of Initiator, briefly swirl

Add 6 drops of Enhancer, briefly swirl

Incubate on shaker for 5-10 minutes

Wash

Carefully pipet off liquid, add several mL of deionized (DI) water,gently swirl

Pipet out beads and place into 1.5 mL tube, remove excess water

Add ˜1 mL of DI water, gently mixing with pipet, then remove water

Repeat step 2-3 more times

Resuspend beads in DI water

Measure 20 individual beads in PDMS channel

Assay (for High Target Concentration)

Mix Capture Beads and Target in glass vial

1.980 mL of Hyb. Buffer

4 μL Capture Beads

21 μL of ssDNA synthetic Target

Incubate on shaker for 20 minutes (for heating see below)

Pipet and discard liquid

Wash

Add 3 mL of Hybridization Buffer, briefly swirl vial

Allow beads to settle, then pipet off wash liquid

Repeat steps 4a and 4b two more times

Mix Capture Beads/Target with AuNP Probes

Add 1.980 mL of Hybridization Buffer

18.4 μL of AuNP probe

Incubate on shaker for 20 minutes (for heating see below)

Pipet and discard liquid

Wash

Add 3 mL of NO₃ ⁻ Buffer, briefly swirl vial

Allow beads to settle, then pipet off wash liquid

Repeat steps 8a and 8b two more times

Ag development

Add 6 drops of Initiator, briefly swirl

Add 6 drops of Enhancer, briefly swirl

Incubate on shaker for 5-10 minutes

Wash

Carefully pipet off liquid, add several mL of DI water, gently swirl

Pipet out beads and place into 1.5 mL tube, remove excess water

Add ˜1 mL of DI water, gently mixing with pipette, then remove water

Repeat step 2-3 more times

Resuspend beads in DI water

Measure 20 individual beads in PDMS channel

For Heating:

Ensure water bath is up to temperature (e.g. 90° C.) before beginningassay

After adding all reagents to each vial, immerse the bottom portion ofeach vial in water bath and gently swirl the vial(s) for the specifiedheating time (e.g. 90 s)

Note: other samples that are not heated should be on shaker at roomtemperature while heated samples are in water bath; the total incubationtime (20 min) should include the time for heating.

Example 14.3: Measuring SERS Beads

Using Peek Seeker Pro:

100 mW

10 s exposure

50× objective, Olympus microscope

Determine location of focused beam spot within video field of view

2. Each bead should be approximate size of beam spot

3. With video on (and laser off) position single isolated bead in thearea where beam area; turn off light, acquire measurement; laser spotshould make outline of the bead.

DNA Sequences Used

ID Name 5′ −> 3′ BA-Target ACA GAG GGA TTA TTG TTA AAT ATTGAT AAG GAT ATA (SEQ ID No. 3) BA-Probe-StrandTAA CAA TAA TCC CTC TGT [Cy5] [Thiol C3] (SEQ ID No. 4)BA-Capture-Strand [Thiol C6] TAT ATC CTT ATC AAT ATT (SEQ ID No. 5)VEE-Target TGA CAA GAC GTT CCC AAT CAT GTT GGA AGG GAA GAT(SEQ ID No. 6) VEE-Probe Strand ATT GGG AAC GTC TTG TCA [Cy3.5][Thiol C3] (SEQ ID No. 7) VEE-Capture Strand [Thiol C6]ATC TTC CCT TCC AAC ATG (SEQ ID No. 8) YP-TargetAGA GTA GGA TCA TAT ACC CGT TAG ATG CTG CTG GCG TTA (SEQ ID No. 9)YP-Capture Strand [Thiol C6] TAA CGC CAG CAG CAT CTA ACG (SEQ ID No. 10)YP-Probe Strand GGT ATA TGA TCC TAC TCT [Cy3] [Thiol C3] (SEQ ID No. 11)

Example 15

FIG. 23, depicts an apparatus 1010 in which the systems, analysissystems, breadboard analysis systems, or other systems described hereinmay be coupled to in order to assist in automation of the system. Theapparatus 1010 may be embodied in any one of a wide variety of wiredand/or wireless computing devices, multiprocessor computing device, andso forth. As shown in FIG. 23, the apparatus 1010 comprises memory 214,a processing device 202, a number of input/output interfaces 204, anetwork interface 206, a display 205, a peripheral interface 211, andmass storage 226, wherein each of these devices are connected across alocal data bus 210. The apparatus 1010 may be coupled to one or moreperipheral measurement devices (not shown) connected to the apparatus1010 via the peripheral interface 211.

The processing device 202 may include any custom made or commerciallyavailable processor, a central processing unit (CPU) or an auxiliaryprocessor among several processors associated with the apparatus 1010, asemiconductor based microprocessor (in the form of a microchip), amacroprocessor, one or more application specific integrated circuits(ASICs), a plurality of suitably configured digital logic gates, andother well-known electrical configurations comprising discrete elementsboth individually and in various combinations to coordinate the overalloperation of the computing system.

The memory 214 can include any one of a combination of volatile memoryelements (e.g., random-access memory (RAM, such as DRAM, and SRAM,etc.)) and nonvolatile memory elements (e.g., ROM, hard drive, tape,CDROM, etc.). The memory 214 typically comprises a native operatingsystem 216, one or more native applications, emulation systems, oremulated applications for any of a variety of operating systems and/oremulated hardware platforms, emulated operating systems, etc. Forexample, the applications may include application specific softwarewhich may be configured to perform some or all of the methods describedherein (Labview, for example). In accordance with such embodiments, theapplication specific software is stored in memory 214 and executed bythe processing device 202. One of ordinary skill in the art willappreciate that the memory 214 can, and typically will, comprise othercomponents which have been omitted for purposes of brevity.

Input/output interfaces 204 provide any number of interfaces for theinput and output of data. For example, where the apparatus 1010comprises a personal computer, these components may interface with oneor more user input devices 204. The display 205 may comprise a computermonitor, a plasma screen for a PC, a liquid crystal display (LCD) on ahand held device, or other display device.

In the context of this disclosure, a non-transitory computer-readablemedium stores programs for use by or in connection with an instructionexecution system, apparatus, or device. More specific examples of acomputer-readable medium may include by way of example and withoutlimitation: a portable computer diskette, a random access memory (RAM),a read-only memory (ROM), an erasable programmable read-only memory(EPROM, EEPROM, or Flash memory), and a portable compact disc read-onlymemory (CDROM) (optical).

With further reference to FIG. 23, network interface device 206comprises various components used to transmit and/or receive data over anetwork environment. For example, the network interface 206 may includea device that can communicate with both inputs and outputs, forinstance, a modulator/demodulator (e.g., a modem), wireless (e.g., radiofrequency (RF)) transceiver, a telephonic interface, a bridge, a router,network card, etc.). The apparatus 1010 may communicate with one or morecomputing devices via the network interface 206 over a network. Theapparatus 1010 may further comprise mass storage 226. The peripheral 211interface supports various interfaces including, but not limited toIEEE-1394 High Performance Serial Bus (Firewire), USB, a serialconnection, and a parallel connection.

Example 16

Systems as described herein for biological target analysis can beconstructed from a combination of interchangeable elements or modules,as described herein. FIGS. 24A-24D demonstrate a few example schematicshowing embodiments of systems that can be constructed as describedherein. Fluid, such as gas or a liquid, that contains biological targetmolecules can flow through the modules according to the arrowspresented. The examples show serial connection of modules, but multipletypes of an individual module can be used, and serial connections aswell as parallel connections (in some cases serial and parallel) can beutilized as well. Connections to controllers and/or computing devicesare not shown in the FIGS. 24A-24D, but in some embodiments, the system(or modules therein) can have electrical and/or operational connectionsbetween pumps and/or valves within to a controller and/or computingdevice and be configured to receive instructions from the controllerand/or computing device.

Example 17

Described herein are systems for detection of biologic targets thatutilize interchangeable modules. FIGS. 25A-25F illustrate exampleconfigurations of basic, generic configurations that can be utilized toconfigure modules as described herein. As it's most basic, a module canhave a sample chamber for sample processing as shown in FIGS. 25A-25F. Asample chamber can be any size or shape that can hold a sample in afluid, such as a gas or liquid. A sample chamber can be a glasscapillary tube, a plastic centrifuge tube (such as an Eppendorf 1.5 mLtube), Teflon tubing, and can contain a filter or a membrane, such as a20 μm filter or a silica membrane. A sample chamber can hold biologicalsamples to be processed, and can provide an volume where samples can bemixed with reagents in the module.

Reagents in the module can be stored in reagent vessels, chambers, orloops. Although reagent vessels are shown in FIGS. 25A-25F, a reagentvessel can also be a reagent loop, and can be a plastic container, suchas a plastic bottle, or can be plastic tubing. Reagent storage vesselsor loops can be in fluidic communication with a sample chamber by way ofplastic tubing or Teflon tubing. Valves (one or more way valves) candirect or restrict fluid communication between a reagent storage vesselor loop and a sample chamber. Valves can be operated manually by anoperator, or can be in electrical/operational connection with acontroller and/or computing device for automated use. Fluid flow from areagent vessel to a sample chamber can be driven by positive fluidpressure with the vessel or loop, or be driven by negative fluidpressure outside of a vessel or loop. Fluid pressure can be manipulatedby one or more pumps, examples of which are described previously. Pumpscan be operated manually or can be electrically and/or operationallycoupled to a controller and/or computing device for automated use.

FIG. 25A shows a reagent vessel connected in series with a samplechamber (in parallel with the input/output fluid path to/from themodule). Other configurations can be realized as shown in FIGS. 25B and25C. Specific configuration of a module can be accomplished by oneskilled in the art according to a desired lysis method and/or targetmolecule and isolation protocol.

As previously mentioned, fluid control valves can be used to direct orrestrict fluid between individual components within a model. FIGS.25D-25F show example configurations of modules that employ valves, andvalves connected to a controller and/or computing device.

FIG. 26 shows an embodiment of a cell lysis module in fluid connectionwith a biological target purification module. The embodiment of the celllysis module is configured for thermal lysis, although otherconfigurations can be realized. The embodiment of the biological targetmodule is an embodiment wherein the configuration of the module is basedon DNA extraction with a Qiagen DNeasy® kit and Qiagen DNeasy®extraction protocol. Other configurations can be realized according tothe specific target molecule of interest and isolationmethodology/protocol for that target. The module embodiment in FIG. 26also employs optional optical sensors which can be used for manual orautomated monitoring of the module during use i.e. procession of targetpurification. Optional electrical/operational connections to acontroller are also shown for automated use, although these can beomitted for a manually operated module in areas where electrical powermay not be readily available.

FIG. 27 shows an embodiment of an assay mixing module according to thepresent disclosure. The configuration of the module utilizes reagentloops, and the module is configured for hybridization of biologicaltarget DNA to microbeads containing capture DNA and nanoparticlescoupled to probe DNA through a spectroscopic label configured for SERSdetection. The embodiment of FIG. 27 incorporates a thermal cyclingheating element for efficient DNA hybridization, and can incorporate anelement that injects gas into the assay tube to create bubbling, whichcan be beneficial for the mixing of reagents in the assay tube. Otherconfigurations of this module can be realized according to the presentdisclosure by one skilled in the art according to a desired targetmolecule and detection or analysis procedure. Example 14 shows examplesof assays the assay mixing module or other modules of a system could beconfigured to carry out.

Although now shown in FIGS. 25A-25F, pumps can be used to drive fluidswithin a module or between modules in the system. One or more pumps canbe in fluid connection with a module via the input or output shown inFIGS. 25A-25F, and can create negative or positive pressure. Pumps canalso be in fluid connection with or integrated into reagent vessels todrive reagent movement throughout a module. Examples of pumps that canbe used can be syringe pumps, syringes, di-electrophoresis pumps, andsolid propellant pumps, although other pumps known in the art can alsobe used. Pumps can be manually operated or can be operationally coupledto a controller and/or computing device via standard electronicconnections for automated use.

Unless defined otherwise, all technical and scientific terms used havethe same meaning as commonly understood by one of ordinary skill in theart to which this disclosure belongs. Although any methods and materialssimilar or equivalent to those described can also be used in thepractice or testing of the present disclosure, the preferred methods andmaterials are now described.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of separating, testing, and constructingmaterials, which are within the skill of the art. Such techniques areexplained fully in the literature.

It should be emphasized that the above-described embodiments are merelyexamples of possible implementations. Many variations and modificationsmay be made to the above-described embodiments without departing fromthe principles of the present disclosure. All such modifications andvariations are intended to be included herein within the scope of thisdisclosure and protected by the following claims.

At least the following us claimed:
 1. A system for manipulating fluidscomprising: an assay mixing module comprising a plurality of microbeadcomplex components contained in one more reagents to generate one ormore non-stationary microbead complexes, wherein the assay mixing moduleis configured to mechanically and thermally mix isolated biologicaltargets with a plurality of microbead complex components, a flow path influidic communication with the assay mixing module and configured toreceive the one or more non-stationary microbead complexes; an analysisregion comprising a microfluidic glass channel or microfluidic capillaryconfigured to analyze the one or more non-stationary microbead complexeswith a spectrometer; a biological target purification module configuredto receive products of cell lysis and isolate biological targets withone or more silica membranes or capillaries, wherein the biologicaltarget purification module comprises one or more reagent vessels;wherein the biological target purification module is in fluidiccommunication with the assay mixing module and configured to sendisolated biological targets to the assay mixing module; a sample lysismodule configured to receive one or more biological samples, wherein thesample lysis module is configured for thermal or enzymatic lysis to lysecells and create products of cell lysis, the products of cell lysiscomprising biological targets, wherein the sample lysis module isconfigured to send biological targets to the biological targetpurification module; and a sample isolation module containing one ormore filters with micro-scale pores configured to isolate biologicalsamples of a desired size, wherein the sample isolation module is influidic communication with the cell lysis module, wherein the sampleisolation module is configured to receive samples containing biologicalsamples from outside of the device and configured to send biologicalsamples of a desired size to the cell lysis module.
 2. The system ofclaim 1, wherein the plurality of microbead complex components comprise:one or more microbeads with one or more capture molecules coupledthereto; one or more nanoparticles with one or more probe moleculescoupled thereto by way of a label; and wherein the capture molecule andprobe molecule are configured to bind to one or more isolated biologicaltargets.
 3. The system of claim 2, wherein the label is a spectroscopicdye.
 4. The system device of claim 2, wherein the one or more microbeadsis at least two orders of magnitude larger than the one or morenanoparticles.
 5. The system device of claim 2, wherein the one or moremicrobeads and one or more nanoparticles are within respective separatesolutions or are contained within a common solution.
 6. The system ofclaim 2, further comprising: at least two microbeads having at least twodifferent respective capture molecules coupled thereto; and at least twonanoparticles having at least two different respective probe moleculescoupled thereto via different respective labels, the differentrespective capture molecules and different respective probe moleculesconfigured to be, respectively, coupled together via differentrespective biological targets to form a random array of at least twonon-stationary microbead complexes in random locations in a fluid. 7.The system of claim 2, wherein the label is configured to provide aRaman spectrum to a portable Raman spectrometer.
 8. The system of claim2, wherein the capture molecule is a DNA or RNA molecule configured tobind to a biological target of a biowarfare agent.
 9. The system ofclaim 2, wherein at least one of the one or more capture molecules andone or more probe molecules is an aptamer, antibody, or nucleic acid.10. The system of claim 2, wherein the one or more microbeads havemultiple capture molecules coupled thereto, wherein the multiple capturemolecules are the same or different.
 11. The system of claim 2, whereinthe nanoparticle is a gold nanoparticle.
 12. The system of claim 11wherein the gold nanoparticle has one or more silver nanoparticlesattached thereto.
 13. The system of claim 1, wherein the systemcomprises one or more valves operatively coupled to a controller, acomputing device, or both, wherein the one or more valves are configuredto direct or restrict flow between the modules of the system, vesselswithin a module, or both.
 14. The system of claim 1, further comprisingone or more pumps operationally coupled to the system and configured toprovide positive fluid pressure, negative fluid pressure, or both in thesystem.
 15. The system of claim 14, wherein the one or more pumps areoperatively coupled to a controller, a computing device, or both. 16.The system of claim 14, wherein the one or more pumps are syringe pumps,dielectrophoresis pumps, solid chemical propellant pumps, individuallyor in combination.
 17. The system of claim 1, wherein the fluids arefemtofluids, picofluids, nanofluids, or microfluids.
 18. The system ofclaim 1, wherein the isolated biological targets are nucleic acids. 19.The system of claim 1, wherein the one or more non-stationary microbeadcomplexes have a maximum dimension of a size to fit within a focal pointor diameter of the cross-sectional area of a laser beam configured toanalyze the bead complex.